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.     

Free and erythrocyte-bound bilirubin in neonatal jaundice

Serum free bilirubin and erythrocyte-bound bilirubin concentrations were estimated in 53 jaundiced neonates with a total serum bilirubin concentration of 227.4 +/- 80.4 mumol/l. The serum free bilirubin and erythrocyte-bound bilirubin concentrations were 8.7 +/- 5.6 nmol/l and 23.8 +/- 6.0 mumol/l, respectively. Both, the serum free bilirubin (r = 0.741, p less than 0.001) and erythrocyte-bound bilirubin (r = 0.754, p less than 0.001) correlated directly with bilirubin-albumin molar ratio. There was a direct correlation between free and erythrocyte-bound bilirubin concentration (r = +0.657, p less than 0.001). The prediction of serum free bilirubin concentrations from the known erythrocyte-bound bilirubin value was unreliable.     

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.     

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.     

Management of hyperbilirubinemia and prevention of kernicterus in 20 patients with Crigler-Najjar disease

We summarize the treatment of 20 patients with Crigler-Najjar disease (CND) managed at one center from 1989 to 2005 (200 patient-years). Diagnosis was confirmed by sequencing the UGTA1A gene. Nineteen patients had a severe (type 1) phenotype. Major treatment goals were to maintain the bilirubin to albumin concentration ratio at <0.5 in neonates and <0.7 in older children and adults, to avoid drugs known to displace bilirubin from albumin, and to manage temporary exacerbations of hyperbilirubinemia caused by illness or gallstones. A variety of phototherapy systems provided high irradiance over a large body surface. Mean total bilirubin for the group was 16+/-5 mg/dl and increased with age by approximately 0.8 mg/dl per year. The molar ratio of bilirubin to albumin ranged from 0.17 to 0.75 (mean: 0.44). The overall non-surgical hospitalization rate was 0.12 hospitalizations per patient per year; one-half of these were for neonatal hyperbilirubinemia and the remainder were for infectious illnesses. Ten patients (50%) underwent elective laproscopic cholecystectomy for cholelithiasis. No patient required invasive bilirubin removal or developed bilirubin-induced neurological damage under our care. Visual acuity and color discrimination did not differ between CND patients and age-matched sibling controls. Four patients treated with orthotopic liver transplantation were effectively cured of CND, although one suffered significant transplant-related complications.Conclusions. While patients await liver transplantation for CND, hyperbilirubinemia can be managed safely and effectively to prevent kernicterus. Lessons learned from CND can be applied to screening and therapy of non-hemolytic jaundice in otherwise healthy newborns.     

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.     

Blood-brain barrier permeability to bilirubin in the rat studied using intracarotid bolus injection and in situ brain perfusion techniques

It has generally been assumed that free unconjugated bilirubin gains access to the brain because of its lipid solubility. However, no measurements of the blood-brain barrier permeability to free bilirubin exist. The aim of these experiments was to determine the blood-brain barrier permeability in the rat to free bilirubin using single-pass (Oldendorf) and in situ perfusion (Takasato) techniques. Studies were performed on adult rats under sodium pentobarbitone anesthesia. [3H]bilirubin IX-alpha-ZZ (sp act 6.2 Ci/mmol) was synthesized by reduction of biliverdin with sodium boro-[3H]hydride. Blood-brain barrier permeability to albumin-bound and free bilirubin was determined using injectates/perfusates containing a molar excess of human serum albumin with or without the addition of the displacing agent sulphadimethoxine. For free bilirubin, the brain uptake index was 28.5 +/- 9.3 (mean +/- SD, n = 18), and the permeability surface area product was 54.84 +/- 36.38 x 10(-4) mL/s/g (mean +/- SD, n = 13). These results demonstrate that the behavior of free bilirubin in vivo in relation to the cerebral microvasculature corresponds to that of a "lipid soluble" molecule.     

The effects of bilirubin on brain energy metabolism during normoxia and hypoxia: an in vitro study using 31P nuclear magnetic resonance spectroscopy

Available evidence from in vitro studies suggests that the neurotoxic effects of bilirubin may be exacerbated by, or even require, additional factors such as hypoxia or asphyxia. The aim herein was to use 31P nuclear magnetic resonance spectroscopy to study the effects of bilirubin on brain energy metabolism in vitro under conditions of normoxia and hypoxia. 31P nuclear magnetic resonance spectra were acquired from guinea pig cerebral hemisphere slices during superfusion with solutions containing bilirubin and albumin in 5:1 molar ratio. The effects of bilirubin at concentrations between 400 nmol/liter and 120 mumol/liter were studied under normoxic conditions. Bilirubin caused no apparent disruption in brain energy metabolism during normoxia. The combined effects of bilirubin (40 mumol/liter) and hypoxia were studied. Hypoxia alone led to a steady state reduction in the phosphocreatine to inorganic phosphate peak-height ratio to 0.30 (0.27-0.32) [mean (range) n = 3]. Bilirubin (40 mumol/liter) in the presence of hypoxia caused a further reduction in the phosphocreatine to inorganic phosphate ratio to 0.18 (0.17-0.20) [mean (range) n = 3, p less than 0.01, analysis of variance] which was rapidly reversed on returning to normoxia. These results demonstrate that bilirubin at the concentration studied requires hypoxia, in addition, to cause a measurable disturbance of brain energy metabolism. The nature of this interaction is unknown, but it may reflect the effect of intracellular acidosis on bilirubin solubility or the oxygen dependence of brain mitochondrial bilirubin oxidase.     

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.     

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.     

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.     

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.     

The pathochemistry of kernicterus.

The stoichiometry of bilirubin--albumin interaction has been analyzed and quantitated in several recent studies, confirming that albumin binding of bilirubin obeys the law of mass action [4, 5, 14, 16, 26, 36, 43, 46, 61, 65, 73, 92, 111]. These studies provide a basis for interpreting bilirubin transport, cell uptake and toxicity from physicochemical and pharmacologic perspectives [35, 42, 58, 59]. In this report, we propose a model of the pathogenesis of kernicterus which views serum albumin and tissue as competing with each other for binding the miscible bilirubin pool. Evidence is presented to show that bilirubin normally binds reversibly to cellular membranes and certain soluble enzymes just as it does to albumin; the unbound bilirubin concentration is the driving force for both albumin and tissue binding. We propose that albumin binding is determined by the concentration of free bilirubin anion (which is essentially unaffected by physiologic pH changes), and that tissue binding is mainly determined by the concentration of free bilirubin acid (which is greatly influenced by pH). When bilirubin--tissue complexes are formed, essential cell functions may be inhibited, producing cellular acidosis, irreversible intracellular aggregation of bilirubin, and cell death. In developing this argument, we will sequentially discuss relevant features of bilirubin chemistry, the binding of bilirubin to albumin, the formation of bilirubin--tissue complexes, bilirubin toxicity, alternative viewpoints of bilirubin transport, and, finally, the implications of this model to the clinical management of jaundiced infants. It should be emphasized that this paper is an attempt to analyze bilirubin transport and toxicity using basic chemical principles; it is an extension of previously published proposals [17, 77], and will undoubtedly require further modification as additional experimental data becomes available.     

Dietary origin of retained H+ in infants with acquired monosaccharide intolerance

Net external acid balance was studied in 12 malnourished infants with chronic diarrhea (some of whom had acquired monosaccharide intolerance). When the infants achieved an adequate energy intake from a formula that contained either glucose or glucose polymers, seven developed metabolic acidosis and five remained free of acidosis. During the study, the acidotic infants produced a significant excess of acid (3.7 +/- 2 vs. 0.5 +/- 2 mEq/kg/day, p less than 0.005). The amount they excreted in urine (2.9 +/- 2 mEq/kg/day), however, was similar to that excreted by nonacidotic infants (2.7 +/- 2 mEq/kg/day) and indicated renal inability to reduce the excess acid load. The net effect was hydrogen ion (H+) retention (+0.8 +/- 0.8 vs. -2.2 +/- 0.8 mEq/kg/day, p less than 0.001). Good correlation existed between the net acid balance and the acid-base measurement in the blood. We speculate that (a) the increased acid load was a consequence of colonic bacterial production of volatile fatty acids from carbohydrate malabsorbed from the small bowel and (b) the renal incapacity to excrete H+ probably was secondary to potassium and phosphate depletion.     

Red Cell Membrane Lipid Peroxidation and Hemolysis Secondary to Phototherapy

ABSTRACT. The exposure of red cells to phototherapy light in the presence of a sensitizer (bilirubin) resulted in oxidative injury to the red cell membrane as manifested by a significant increase in the concentration of the products of lipid peroxidation (TBA reactants and diene conjugation) in the membrane and hemolysis. To induce a photo-oxidized membrane injury, the sensitizer (bilirubin) has to be membrane bound. Thus, by altering the availability of free bilirubin in the red cell suspension through changes in the molar concentration ratio of bilirubin to albumin, one is able to regulate the occurrence and extent of the oxidative red cell membrane injury. The clinical implications of these findings are discussed.     

Red cell membrane lipid peroxidation and hemolysis secondary to phototherapy

The exposure of red cells to phototherapy light in the presence of a sensitizer (bilirubin) resulted in oxidative injury to the red cell membrane as manifested by a significant increase in the concentration of the products of lipid peroxidation (TBA reactants and diene conjugation) in the membrane and hemolysis. To induce a photo-oxidized membrane injury, the sensitizer (bilirubin) has to be membrane bound. Thus, by altering the availability of free bilirubin in the red cell suspension through changes in the molar concentration ratio of bilirubin to albumin, one is able to regulate the occurrence and extent of the oxidative red cell membrane injury. The clinical implications of these findings are discussed.     

BILIRUBIN, RESERVE ALBUMIN FOR BINDING OF BILIRUBIN AND pH IN PLASMA DURING PHOTOTHERAPY (ORDINARY AND DOUBLE LIGHT) OF TERM NEWBORN INFANTS

ABSTRACT. Ebbesen, F. (Department of Neonatology, Rigshospitalet, Copenhagen, Denmark). Bilirubin, reserve albumin for binding of bilirubin and pH in plasma during phototherapy (ordinary and double light) of term newborn infants. Acta Paediatr Scand, 70:223, 1981. –Forty-five term newborn infants with uncomplicated hyperbilirubinaemia were treated continuously with phototherapy for 24 hours. Twenty-eight infants received double light treatment and 17 infants ordinary phototherapy. During both treatments a significant decrease in the serum unconjugated bilirubin concentration, a significant increase in the serum reserve albumin concentration for binding of bilirubin determined by the [¹⁴C] MADDS method, and a significant decrease in the index of serum bilirubin toxicity occurred. The changes in these parameters were significantly greater during the double light treatment than during the ordinary phototherapy. During the treatment the fall in index was constant. No significant change in plasma pH was seen. Thus, the study gives further evidence that the risk of bilirubin encephalopathy is reduced by phototherapy and that double light treatment is in the respect superior to ordinary phototherapy. Prior to phototherapy the molar ratio in serum of unconjugated bilirubin plus reserved albumin for binding of bilirubin to albumin was only 0.60, on average, and during the treatment the increase in the serum reserve albumin concentration was less than the decrease in the serum bilirubin concentration. This can be explained either by the presence in infant serum of an unknown ligand interfering competitively or allosterically in the binding of MADDS and bilirubin to albumin, or by the existence of a foetal albumin with a lower affinity for MADDS than adult albumin.     

Bilirubin toxicity in neural cell lines N115 and NBR10A

The toxicity of bilirubin was investigated in 2 neural cell lines NBR10A and N115 using a quantitative dye assay 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium biomide (MTT) as a measure of cell viability and [3H]thymidine incorporation as a measure of DNA synthesis. Short exposures (up to 2 h) to bilirubin, even up to a bilirubin-albumin molar ratio of 1.5, yielded no evidence of toxicity using these assays. At longer exposure times (24 h) a decrease in cell viability and [3H]thymidine incorporation was detected at a molar ratio of 0.8 when the bilirubin concentration was 0.1 mM or higher, whereas lower bilirubin levels at this molar ratio showed no deleterious effect. The effect of bilirubin is more pronounced at a molar ratio of 1.5 with longer incubation periods. The MTT assay showed the N115 cells appeared to be more resistant to bilirubin cytotoxicity than NBR10A cells, a finding which was not obtained from [3H]thymidine incorporation studies. This discrepancy can be explained by the fact that we are measuring two different variables; the MTT assay estimates the number of viable cells at the end of the experiment by measuring mitochondrial function whereas the [3H]thymidine assay measures the rate of DNA synthesis during the last 2 h of the experiment. The concentration effect of bilirubin is evident from the [3H]-thymidine studies in that at a molar ratio of 1.5 and bilirubin concentration of 0.075 mM or higher, there is both cell kill (decrease in DNA) and inhibition of [3H]thymidine incorporation (decrease in specific activity).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of bilirubin on brain energy metabolism during hyperosmolar opening of the blood-brain barrier: an in vivo study using 31P nuclear magnetic resonance spectroscopy

Despite intensive investigation it remains uncertain how bilirubin enters the brain and how it exerts a toxic effect on neurones. Studies of induced hyperbilirubinemia in animal models in vivo have failed to reproduce bilirubin encephalopathy without additional factors such as hypoxia, asphyxia, hypercapnia, and disruption of the blood-brain barrier. The aim of this study was to investigate, using 31P NMRS, whether hyperbilirubinemia alone or in association with hyperosmolar opening of the blood-brain barrier caused any disturbance of cerebral energy metabolism in vivo. Spectra were acquired using a surface coil positioned over the right cerebral hemisphere of anaesthetized adult rats placed in the bore of a 1.9 Tesla magnet. Hyperbilirubinemia alone at a maximum mean serum concentration of 1063 +/- 175 mumol/L (mean +/- SD, n = 7) caused no apparent disruption in brain energy metabolism. However, in combination with hyperosmolar blood-brain barrier opening a serum bilirubin concentration of 483 +/- 52 mumol/L (mean +/- SD, n = 9) was associated with a reduction in PCr/(PCr + Pi) ratio from 0.68 +/- 0.06 to 0.44 +/- 0.14 (mean +/- SD, p less than 0.001). A significant correlation was demonstrated between cerebral hemisphere bilirubin content and the reduction in PCr/(PCr + Pi) (r = 0.84, n = 9, p less than 0.01). These results demonstrate in vivo a disruptive effect of bilirubin on cerebral energy metabolism in the presence of an open BBB. This mode of entry and mechanism of toxicity may be factors in the pathophysiology of bilirubin encephalopathy in the newborn infant.     

Relationship of unbound bilirubin concentration to reserve albumin-binding concentration for bilirubin in human neonatal plasma

We examined the relationship of plasma unbound bilirubin concentration and reserve albumin-binding concentration for bilirubin in a sample of 545 neonates. Plasma unbound bilirubin concentration and total bilirubin-binding concentration were determined with the peroxidase assay. Contrary to published reports, we found that plasma unbound bilirubin concentration and plasma reserve albumin for bilirubin-binding concentration are highly correlated (r = -0.706; p less than 0.001) and that the relationship between these two parameters is dependent upon the total bilirubin-binding concentration. That these measured parameters correlate in the same manner as predicted for free bilirubin and free albumin by the law of mass action, suggests that these measurements may be meaningful.