BILIRUBIN DISPLACING EFFECT OF STABILIZERS ADDED TO INJECTABLE PREPARATIONS OF HUMAN SERUM ALBUMIN

Abstract Stabilizers added to preparations of human serum albumin before heat treatment were tested for bilirubin displacing effect, using the peroxidase method. It was found that N-acetyltryptophan and sodium caprylate displace bilirubin from its complex with human serum albumin in vitro. The quantitative findings were used for a rough estimate of the effect of these substances on the free bilirubin concentration in blood plasma, expected when stabilized albumin preparations are given intravenously for prevention of kernicterus. The calculated effect is a delay of the decrease of free bilirubin concentration, or even a temporary increase. Sodium mandelate displaces less strongly.     

COMPARISON BETWEEN TWO PREPARATIONS OF HUMAN SERUM ALBUMIN IN TREATMENT OF NEONATAL HYPERBILIRUBINAEMIA

ABSTRACT. Thirty-six newborn infants with normal birth weights and with uncomplicated hyperbilirubinaemia, treated with light, were studied. At onset of phototherapy the infants received intravenously 1 g human serum albumin (HSA) per kg body weight as a 9 % solution. Two different preparations of HSA were used and compared. One of these, HSA_I, contained sodium caprylate and N-acetyltryptophan, 5 mmol/1 of each, as stabilizers. HSA_II contained only caprylate, 5 mmol/1. Nineteen infants received HSA_I and seventeen infants HSA_II. The reserve albumin for binding of bilirubin, measured by the [¹⁴C] MADDS method, was low in both preparations in vitro. During the infusion, the serum concentrations of albumin and reserve albumin increased and the serum unconjugated bilirubin concentration decreased, resulting in a fall in the index of plasma bilirubin toxicity in all infants. After completion of the infusion, the serum concentrations of albumin and reserve albumin declined, and a slight rise in index occurred. The increase in the serum reserve albumin concentration was markedly higher during infusion of HSA_II than of HSA_I. It is concluded that infusion of both HSA preparations during phototherapy provides an immediate protection against bilirubin encephalopathy. HSA_I is inferior to HSA_II, probably due to its content of N-acetyltryptophan.     

EFFECTS OF STABILIZERS IN PREPARATIONS OF HUMAN SERUM ALBUMIN ON THE SERUM BILIRUBIN IN GUNN RATS

ABSTRACT. Commercially available preparations of human serum albumin (HSA) containing stabilizers (i.e. 16 mmol/I Na caprylate plus 16 mmol/I Na N-acetyl- dl -tryptophan) were injected either s.c., i.p. or i.v. into homozygous infant Gunn rats. 30 min and 3 hours after s.c. injection, a serum bilirubin decline which surpassed dilution by the injected volume could be ascertained. It was mainly caused by N-acetyl- dl -tryptophan since s.c. injections of appropriate amounts of this substance alone or a mixture of both components of the stabilizer without HSA brought about similar results. HSA without these stabilizers had not such an effect. It is postulated that under these conditions Na N-acetyl- dl -tryptophanate displaced bilirubin from albumin bonds. It became obvious that after s.c. injection equilibration of HSA between skin and plasma was delayed, whereas Na N-acetyl- dl -tryptophan was rapidly transported to the blood. As for Na caprylate, a displacing effect of short duration could not be excluded by the experimental arrangement used, since the metabolism of the substance in the rat is very fast. When HSA and the stabilizers entered the plasma simultaneously (i.v. injection) no effect on serum bilirubin concentration could be proved 30 min and 3 hours later. All the bilirubin and the Na N-acetyl- dl -tryptophan present in the plasma at that time can be bound to the large amount of albumin which is directly given into the circulation of the animal. 30 min after i.p. injection of HSA preparations containing stabilizers a small decrease of serum bilirubin concentration could be recognized. It was less pronounced and less persisting than after s.c. injection. Probably equilibration of HSA between peritoneum and plasma went on faster than between skin and plasma. Only for a short period a lack of albumin binding sites in the plasma of the rat pointed to a surplus of Na N-acetyl- dl -tryptophan.     

Experimental bilirubin encephalopathy: importance of total bilirubin, protein binding, and blood-brain barrier

The cause of bilirubin encephalopathy has been variously ascribed to elevated total serum bilirubin concentration, high free bilirubin levels (or impaired albumin binding), and disruption of the blood-brain barrier. An experimental rat model for acute bilirubin encephalopathy was developed in which these three factors could be varied independently. Osmotic opening of the blood-brain barrier in the right hemisphere was produced by infusing a hypertonic arabinose solution into the right carotid artery. The total bilirubin level and bilirubin binding state were varied by adjusting the amount of bilirubin infused intravenously and/or by infusing human serum albumin. Brain electrical activity (EEG) served as an indicator of developing encephalopathy. Neither staining nor EEG changes occurred if the blood-brain barrier remained intact. Bilirubin staining without EEG evidence of encephalopathy sometimes occurred when the blood-brain barrier was open. Discriminant analysis showed that EEG changes were best predicted by the degree of blood-brain barrier opening (as indicated by brain bilirubin content) and by the quality of serum bilirubin binding. Serum total bilirubin concentration was not an important discriminator of encephalopathy.     

Ceftriaxone effect on bilirubin-albumin binding

The effect of ceftriaxone on bilirubin-albumin binding was measured in vitro using the peroxidase method with human serum albumin and a dialysis rate method with adult and newborn serum. Ceftriaxone competes with bilirubin for binding to human serum albumin; the displacement constant is 1.5 X 10(4) L/mol. Therapeutic levels of ceftriaxone decrease the reserve albumin concentration in newborn serum by 39%. These results indicate that ceftriaxone may increase the risk of bilirubin encephalopathy in jaundiced premature infants.     

Increase in bilirubin binding to albumin with correction of neonatal acidosis.

Twenty-six serial measurements of free bilirubin concentration and apparent association constant of bilirubin for albumin (Ka) at a bilirubin: albumin molar ratio of 0.8 were performed and compared with baseline values in 11 newborn infants with acidosis before treatment and during recovery from acidosis. When arterial pH was corrected from 7.12 +/- 0.02 (Mean +/- S.EM.) to 7.34 +/- 0.02, there was a significant decrease in serum free bilirubin concentration and a significant increase in the Ka at molar ratio 0.8. The data offer in vivo evidence that correction of acidosis in the neonate results in an improvement of the apparent bilirubin binding affinity of albumin.     

INCREASE IN BILIRUBIN BINDING TO ALBUMIN WITH CORRECTION OF NEONATAL ACIDOSIS

Abstract. Twenty-six serial measurements of free bilirubin concentration and apparent association constant of bilirubin for albumin (Ka) at a bilirubin: albumin molar ratio of 0.8 were performed and compared with baseline values in 11 newborn infants with acidosis before treatment and during recovery from acidosis. When arterial pH was corrected from 7.12±0.02 (Mean±S.E.M.) to 7.34±0.02, there was a significant decrease in serum free bilirubin concentration and a significant increase in the Ka at molar ratio 0.8. The data offer in vivo evidence that correction of acidosis in the neonate results in an improvement of the apparent bilirubin binding affinity of albumin.     

The importance of free bilirubin acid salt in bilirubin uptake by erythrocytes and mitochondria

The binding of bilirubin to tissue was studied using adult human erythrocytes and rat liver mitochondria. Tissues were incubated with varying bilirubin-albumin molar ratios, varying albumin concentrations of a given bilirubin-albumin molar ratio, and varying pH. Bilirubin binding by tissue was reversible and stoichiometric with the concentration of the free (nonalbumin bound) bilirubin acid salt (bilirubin monovalent anion). Minimal binding of the bilirubin dianion, the predominant state of bilirubin in plasma, was also suggested. The observations support the "free bilirubin theory" where tissue and albumin compete for binding the body's bilirubin pool. Binding to tissue, however, is not determined by the free bilirubin concentration, but by the concentration of the pH dependent subfraction, the free bilirubin acid salt. Tissue binding and toxicity of bilirubin may result from the surfactant properties of the monovalent anion.     

Effect of cephalosporins on bilirubin-albumin binding

The effect of parenterally administered cephalosporins on bilirubin-albumin binding was measured in vitro by means of the peroxidase method with human serum albumin and by means of a dialysis rate method with newborn infants' serum. Ceftriaxone and cefoperazone have been shown to affect bilirubin-albumin binding. In this study, 13 additional cephalosporins were tested. Cefonicid, cefotetan, and cefmetazole competed with bilirubin for albumin binding and, at reported mean peak serum levels, decreased the reserve albumin concentration by 75%, 56%, and 40%, respectively. We suggest that these five cephalosporins may increase the risk of bilirubin encephalopathy in jaundiced neonates.     

Salicylate saturation index in neonatal jaundice.

30 serum samples from premature and newborn infants with non-haemolytic hyperbilirubinaemia were analyzed to prove the accuracy of determination of albumin binding capacity for bilirubin. The salicylate method of Odell was used to determine the saturation index of albumin indicated by a decrease in optical density through displaced bilirubin. Bilirubin is stoichometrically displaced from albumin by the addition of salicylate. The values of the sautration index correspond to free binding sites. Analysis of our data demonstrated that there is no direct correlation between the saturation index (SI) and total serum bilirubin/albumin concentration quotient. Methodical errors, problems in statistics and other theoretical concepts are discussed. The salicylate method is not suitable for accurate determination of albumin binding capacity for 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.     

Albumin binding properties in relation to bilirubin and albumin concentrations during the first week of life

In 19 non-jaundiced and 22 jaundiced neonates, the serum albumin and bilirubin concentrations were measured during the first week of life. Some of the neonates were followed longitudinally. The albumin binding properties were evaluated by determining the reserve albumin concentration for monoacetyldiaminodiphenyl sulphone (MADDS), a deputy ligand for bilirubin. The reserve albumin concentration for MADDS increased with postnatal age. The reason for this increase is still unexplained. There was an inverse relation between the bilirubin and the reserve albumin concentrations, but when the bilirubin concentration increased by 1 mumol/l, the reserve albumin concentration for MADDS decreased by only 0.2 mumol/l. This shows that the reserve albumin concentration for MADDS does not give a direct measure of the bilirubin binding ability of the serum albumin molecule. In spite of this, it is still possible that a low reserve albumin concentration for MADDS is a risk factor for bilirubin encephalopathy.     

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 lactate, pyruvate, acetone, acetoacetate, and beta-hydroxybutyrate on albumin binding of bilirubin

Lactate, pyruvate, acetone, acetoacetate, and beta-hydroxybutyrate were tested for their bilirubin-displacing effect on human serum albumin. Only lactate had a significant effect at levels found in asphyxiated infants (up to 20 mM). The reserve albumin equivalent for binding bilirubin was determined, using the deputy ligand monoacetyldiaminodiphenyl sulfone (MADDS), in adult human serum albumin solution, neonatal serum, and neonatal albumin solution. Twenty mM lactate caused a 23% decrease of reserve albumin when adult albumin was used, but did not cause any change of binding when neonatal serum or neonatal albumin solution was used. It is unlikely that endogenous substances, acting as competitive ligands, cause the low binding affinity of albumin for bilirubin in sick, premature infants.     

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     

Albumin Binding Properties in Relation to Bilirubin and Albumin Concentrations during the First Week of Life

ABSTRACT. In 19 non-jaundiced and 22 jaundiced neonates, the serum albumin and bilirubin concentrations were measured during the first week of life. Some of the neonates were followed longitudinally. The albumin binding properties were evaluated by determining the reserve albumin concentration for monoacetyldiaminodiphenyl sulphone (MADDS), a deputy ligand for bilirubin. The reserve albumin concentration for MADDS increased with postnatal age. The reason for this increase is still unexplained. There was an inverse relation between the bilirubin and the reserve albumin concentrations, but when the bilirubin concentration increased by 1 μmol/l, the reserve albumin concentration for MADDS decreased by only 0.2 μmol/l. This shows that the reserve albumin concentration for MADDS does not give a direct measure of the bilirubin binding ability of the serum albumin molecule. In spite of this, it is still possible that a low reserve albumin concentration for MADDS is a risk factor for bilirubin encephalopathy.     

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.     

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.     

The effect of bilirubin on the surface charge and aggregation tendency of platelets in cord blood

Unbound unconjugated bilirubin markedly increased the negative electrophoretic mobility of washed platelets from cord blood, even at concentration as low as 1.5 mg/dl. The increase in negativity could be abolished by washing the platelets. Aggregation of platelets was observed in parallel with the increase in negativity. These actions of bilirubin required calcium ions, which could not be replaced by magnesium ions. The results suggest attachment of the negative bilirubin molecules onto the platelet surface, probably through calcium ions, leading to platelet aggregation.Plasma components inhibited the actions of bilirubin on platelets up to 19.5 mg/dl bilirubin concentration.The effect of bilirubin on platelets was also investigated in the presence of albumin. When the saturation of albumin with bilirubin was exceeded, platelet negativity increased. However, the fraction of free bilirubin exceeding the albumin saturation point has not the same effect as truly free bilirubin at that concentration in the complete absence of albumin. The results indicate that albumin molecules loosly adsorbed onto the surface may protect the platelets against the attachment of free bilirubin. This protection, however, might be impaired by acidosis, which is frequently combined with hyperbilirubinaemia in the sick newborn. It is suggested that bilirubin could contribute to the haemostatic abnormalities in sick babies by acting on the platelet surface.