Postnatal changes in the ability of plasma albumin to bind bilirubin

The plasma concentrations of total albumin, unconjugated bilirubin and reserve albumin for bilirubin binding were determined in 407 healthy infants of various age up to eight days. The albumin reserve was measured using monoacetyldiaminodiphenyl-sulfone (MADDS) as a deputy ligand for bilirubin. The fraction of albumin capable of binding bilirubin was calculated as the sum of the concentrations of bilirubin and reserve albumin, divided by the total albumin concentration. Our data showed that this fraction was low (average 0.36) and did not change during the first 24 hours of life, and in this period it was independent of the maturity of the infant, as expressed by its birth weight or gestational age. From about 24 hours of life, the fraction began to increase. This increase came to an end about 60 hours after birth, and no further changes were seen during the following five days. The level of the bilirubin-binding fraction reached 60 hours after birth was related to the maturity of the infant: It increased with increasing birth weight up to 3000 g and with increasing gestational age up to 275 days, when on an average it was about 0.58. The fraction of binding albumin was independent of the sex.     

Benzyl alcohol, kernicterus, and unbound bilirubin

A prospective study in 1983 demonstrated no significant relationship between serum unbound bilirubin levels and kernicterus. The presence of benzoate (a bilirubin-binding competitor) in the serum along with sample dilution, however, may have rendered the unbound bilirubin measurements in that study inaccurate.     

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.     

Reduced albumin binding of MADDS--a measure for bilirubin binding--in sick children

The reserve albumin concentration for binding of MADDS (monoacetyldiaminodiphenyl sulphone) in plasma is used as a measure of the reserve albumin concentration for binding of unconjugated bilirubin. The aim of the present study was to investigate whether a reduction in the reserve albumin concentration for binding of MADDS could exist in sick children after 5 months of age, where the bilirubin binding properties of the albumin has reached the adult level. The material included 75 children, 1-15 years of age with mild infections, severe bacterial infections, acute viral hepatitis, chronic hepatic diseases or uraemia, and a control group of 22 healthy children. The reserve albumin concentration was significantly lower in children with severe bacterial infections, acute viral hepatitis, and uraemia, than in healthy children (p less than 0.01), while the reserve albumin concentration in children with mild infections and chronic hepatic diseases did not differ significantly from that of the control group (p greater than 0.05). The total albumin concentration in plasma in either of the groups of sick children did not differ significantly from that of the healthy children. The plasma concentration of unconjugated bilirubin was elevated in the group of children with acute viral hepatitis, but not enough to influence the concentration of reserve albumin for binding of MADDS to a significant degree. The reserve albumin concentration was significantly lower in children with acute viral hepatitis than in children with severe bacterial infections (p less than 0.05).     

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.     

Cytotoxicity is predicted by unbound and not total bilirubin concentration

Although it has been suggested that the unbound, free, (B(f)) rather than total (B(T)) bilirubin level correlates with cell toxicity, direct experimental evidence supporting this conclusion is limited. In addition, previous studies never included a direct measurement of B(f), using newer, accurate methods. To test "the free bilirubin hypothesis", in vitro cytotoxicity was assessed in four cell lines exposed to different B(f) concentrations obtained by varying B(T)/Albumin ratio, using serum albumins with different binding affinities, and/or displacing unconjugated bilirubin (UCB) from albumin with a sulphonamide. B(f) was assessed by the modified, minimally diluted peroxidase method. Cytotoxicity varied among cell lines but was invariably related to B(f) and not B(T). Light exposure decreased toxicity parallel to a decrease in B(f). In the absence of albumin, no cytotoxicity was found at a B(f) of 150 nM whereas in the presence of albumin a similar B(f) resulted in a 40% reduction of viability indicating the importance of total cellular uptake of UCB in eliciting toxic effect. In the presence of albumin-bound UCB, bilirubin-induced cytotoxicity in a given cell line is accurately predicted by B(f) irrespective of the source and concentration of albumin, or total bilirubin level.     

POSTNATAL CHANGES IN THE ABILITY OF PLASMA ALBUMIN TO BIND BILIRUBIN

ABSTRACT. The plasma concentrations of total albumin, unconjugated bilirubin and reserve albumin for bilirubin binding were determined in 407 healthy infants of various age up to eight days. The albumin reserve was measured using monoacetyldiaminodiphenyl-sulfone (MADDS) as a deputy ligand for bilirubin. The fraction of albumin capable of binding bilirubin was calculated as the sum of the concentrations of bilirubin and reserve albumin, divided by the total albumin concentration. Our data showed that this fraction was low (average 0.36) and did not change during the first 24 hours of life, and in this period it was independent of the maturity of the infant, as expressed by its birth weight or gestational age. From about 24 hours of life, the fraction began to increase. This increase came to an end about 60 hours after birth, and no further changes were seen during the following five days. The level of the bilirubin-binding fraction reached 60 hours after birth was related to the maturity of the infant: It increased with increasing birth weight up to 3000 g and with increasing gestational age up to 275 days, when on an average it was about 0.58. The fraction of binding albumin was independent of the sex.     

The risk of bilirubin encephalopathy, as estimated by plasma parameters, in neonates strongly suspected of having sepsis

The study comprises 18 mature newborns, strongly suspected of having sepsis, and a control group of 18 mature, healthy newborns with the same postnatal age. The object of the investigation was to compare the risk of development of bilirubin encephalopathy between the two groups, as estimated by plasma parameters. The sepsis group had significantly lower reserve albumin concentration for binding of MADDS ( p <0.01) and significantly lower total albumin concentration ( p <0.01). No significant differences were observed in unconjugated bilirubin concentration and plasma pH. It is suggested that mature newborns with sepsis have a slightly increased risk of developing bilirubin encephalopathy.     

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.     

Skin colour and bilirubin in neonates.

The correlation between the yellow colour of the skin and serum bilirubin concentration, reserve albumin concentration, and pH was investigated in 76 icteric neonates. Significant linear correlation existed between yellow colour of the skin and serum bilirubin concentration, reciprocal of the reserve albumin concentration, and the squared hydrogen ion concentration. Furthermore, the basic yellowness of the skin at birth correlated linearily with the yellow colour of the skin measured when the child became jaundiced. The results support the proposed hypothesis that bilirubin is transferred from plasma to skin through two different mechanisms: (a) leakage of bilirubin-albumin complexes into extravascular spaces and (b) precipitation of bilirubin acid in phospholipid membranes. The latter mechanism suggests that measurement of the yellow colour of the skin may be a better predictor of brain damage than the serum bilirubin concentration and thus be of clinical utility. Measurement of the yellow colour of the skin as a method of obtaining serum bilirubin concentration is unreliable.     

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.     

The possible risk of bilirubin encephalopathy as predicted by plasma parameters in neonates with previous severe asphyxia

The study group consisted of nine mature newborn infants with a previous history of severe asphyxia and a control group of 18 mature, healthy newborns with the same postnatal age and sex. The object of the investigation was to compare the possible risk of development of bilirubin encephalopathy between the two groups as estimated by plasma parameters. The asphyxia group had a significantly lower reserve albumin concentrations for binding of monoacetyldiaminodiphenyl sulphone (P=0.008), a measure of binding of unconjugated bilirubin, and significantly lower total albumin concentrations (P=0.02). No significant difference was observed in unconjugated bilirubin concentration. It is suggested that mature newborns with previous severe asphyxia are at a slightly increased risk of developing bilirubin encephalopathy over and above the well-known risk associated with increased permeability of the blood brain barrier.     

Auditory brainstem response and unbound bilirubin in jaundiced (jj) Gunn rat pups

The role of plasma bilirubin-albumin binding in the pathogenesis of kernicterus in human newborns is controversial. Kernicterus in the jaundiced (jj) Gunn rat pup, an animal model for kernicterus, prolongs interwave intervals and decreases wave amplitude in the auditory brainstem response (ABR). Plasma total bilirubin concentration (TBC), albumin concentration, and unbound bilirubin concentration (UBC), a measure of bilirubin-albumin binding, were measured in 16-day-old jj Gunn rat pups (n = 21) and compared with ABR wave latencies, interwave intervals, and wave amplitudes by linear correlation. The UBC, but not the TBC or TBC/albumin ratio, correlated positively and significantly with ABR I-II and I-III interwave intervals (r = 0.55, p = 0.009, and r = 0.60, p = 0.004, respectively). The UBC, but not the TBC or TBC/albumin ratio, predicts bilirubin toxicity, as measured by bilirubin-induced ABR changes in jj Gunn rat pups.     

Risk of bilirubin acid precipitation in preterm infants with respiratory distress syndrome: considerations of blood/brain bilirubin transfer equilibrium

Twenty-six preterm infants with respiratory distress syndrome (RDS), were examined daily during the first 6 days of life. Twenty-six equally preterm but clinically well infants served as controls. In the RDS infants, plasma albumin concentration was low, hyperbilirubinemia was prolonged, plasma pH was decreased during the first two days, and the concentration of reserve albumin for binding of monoacetyldiaminodiphenylsulfone (MADDS), a deputy ligand for bilirubin, was decreased on the second throughout the sixth day, when compared with the controls. These factors concur in increasing the likelihood of bilirubin acid precipitation in RDS above the increased risk present in preterm infants. The plasma of the preterm controls was supersaturated with respect to crystalline bilirubin acid by an average factor 5 (index of plasma bilirubin toxicity = 0.7) on the first day of life, peaking at a factor 10 (index 1.0) on the third and fourth days while these factors were 10 and 20 (index 1.0 and 1.3), respectively, in the RDS infants. Two of the latter surpassed a level of 60 times supersaturation (index 1.8) where acute precipitation of amorphous bilirubin acid becomes possible.     

Hematin and bilirubin binding to human serum albumin and newborn serum

In jaundiced newborn infants, hemolytic disease is considered a risk factor for kernicterus due to the suspected competition between bilirubin and other hemoglobin breakdown products for albumin binding. We have studied the effect of hematin on bilirubin-albumin binding using the peroxidase assay and a light-scattering technique for measuring unbound bilirubin. Our results show that hematin does not affect bilirubin-albumin binding. To determine if other albumin binding functions are affected by hematin, we used a microdialysis rate technique employing two ligands, diazepam and monoacetyldiaminodiphenyl sulfone (MADDS). Hematin does not utilize the diazepam binding function of albumin, but does decrease the albumin binding of MADDS. The results of this study indicate that the MADDS and bilirubin binding functions are not identical. The clinical usefulness of reserve albumin equivalent determination using MADDS is discussed.     

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.     

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.

     

LOW RESERVE ALBUMIN FOR BINDING OF BILIRUBIN IN NEONATES WITH DEFICIENCY OF BILIRUBIN EXCRETION AND BRONZE BABY SYNDROME

ABSTRACT. The plasma reserve albumin concentration for binding of bilirubin was found to be low in four newborn infants with deficiency of bilirubin excretion, of whom two had the bronze baby syndrome. Thus, the risk of bilirubin encephalopathy was increased. Also the ratio of binding fraction of albumin, i. e. unconjugated bilirubin plus reserve albumin, to total albumin was low. Possible causes of the low reserve albumin concentration and the ratio are discussed.     

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.     

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.