Gerard B. Odell

The dissociation of bilirubin from albumin and its clinical implications

Summary The distribution of bilirubin in the body has been described in terms of diffusion equilibria and competitive binding of tissue elements for the circulating bilirubin which is chemically dissociated from albumin. The supposition was made that only dissociated bilirubin can gain access to intracellular fluid by permeation of cell membranes. The amount of bilirubin diffusing into cell fluid is then dependent upon the concentration gradient of dissociated bilirubin across cell membranes rather than total bilirubin concentration. It was shown that the dissociated bilirubin concentration can be increased in extracellular fluids independently of total bilirubin concentration by reducing the binding capacity of plasma proteins for bilirubin. This situation occurs clinically (1) in premature babies who have a relative hypoalbuminemia, and (2) in some newborn infants whose plasma contains substances which reduce the bilirubin binding capacity of albumin. Such substances are hematin, sulfonamides, salicylate, caffeine sodium benzoate, and increased hydrogen ion concentration. By increasing the circulating albumin concentration, it was shown that the administered albumin will increase the total plasma bilirubin concentration by causing a shift of bilirubin from the extravascular tissues into the plasma. The potential therapeutic value of such a procedure was discussed but requires further study before it can be recommended.

Studies in kernicterus. III. The saturation of serum proteins with bilirubin during neonatal life and its relationship to brain damage at five years

Psychometric studies were performed on a group of 5-year-old children who had been jaundiced during neonatal life. On the basis of the test results, 14 children were considered normal and 18 were classified as having brain damage. The presence or absence of brain damage was compared with clinical and laboratory determinations that had been made on the children during their period of neonatal hyperbilirubinemia. No significant correlations were found between the presence or absence of brain damage and the maximum bilirubin concentration, birth weight, sex, presence or absence of hemolytic disease, or the use of exchange transfusion. A significant correlation was found between the presence or absence of brain damage and the saturation of the serum proteins with bilirubin during infancy.

The influence of bicarbonate administration on blood pH in a “closed system”: Clinical implications

An in vitro comparison was made of the effect of isotonic and hypertonic NaHCO 3 solutions on blood pH when the elimination of carbon dioxide was prevented. The results demonstrate that the rise in P co 2 which occurs after addition of isotonic NaHCO 3 offsets the increase in bicarbonate concentration, and the rise in pH is extremely small. The addition of bicarbonate in hypertonic solutions produces a greater elevation of P co 2 and blood pH may actually fall. The acidosis associated with hypertonicity of extracellular fluids results from the dilution of extracellular buffers and the release of protons from intracellular buffers, such as hemoglobin, in response to the increase in ionic strength.

Studies in kernicterus. II. The determinationof the saturation of serum albumin with bilirubin

A method is described for estimating the relative concentration of free bilirubin in serum by measurement of the saturation of the carrier, albumin, for bilirubin. The technique only requires the determination of the change in optical density at 460 mμ (Δ O.D. 460 mμ) of the diluted serum after addition of a standardized amount of salicylate. The ΔO.D. 460 mμ was calibrated to the per cent decrease in protein-bound bilirubin by separating and chemically determining the free and the remaining protein-bound bilirubin. The technique was applied to hyperbilirubinemic sera from infants in the neonatal period. Infants without hemolytic disease showed a direct correlation between the saturation of their albumin with bilirubin and the bilirubin-protein concentration ratio of the serum. Infants with hemolytic disease did not show such a correlation until after they had had an exchange transfusion. The determination of the free bilirubin offers a more precise means of selecting infants at risk to bilirubin encephalopathy.

The Bronze Baby Syndrome: A Complication of Phototherapy.

An intense grey-brown discoloration of the skin, serum, and urine, and anemia occurred in a premature infant when phototherapy was used to reduce hyperbilirubinemia. Pre-existing hepatic disease was suspected as a cause of the joundice and may have prevented the biliary excretion of the photooxidation products of bilirubin; their retention resulted in the bronze discoloration. A disproportionately high saturation of the serum albumin with bilirubin was observed, suggesting that the retained pigments may compete with bilirubin for protein binding or that the phototherapy altered the capacity of albumin to bind bilirubin.

The distribution of bilirubin between albumin and mitochondria

Mitochondria suspended in aqueous solutions containing albumin and bilirubin can preferentially sequester bilirubin from the aqueous phase of such suspensions. The association of bilirubin with mitochondria is favored when the molar ratio of bilirubin to albumin is above 1. The rate of association of bilirubin with mitochondria is increased in hypertonic media or after the addition of organic anions which compete with bilirubin for protein binding. The concentration ratio of bilirubin to albumin at which bilirubin distribution exceeds the albumin space in vitro corresponds to in vivo concentrations of bilirubin and albumin of 20 mg. and 3 Gm. per 100 ml. of plasma, respectively.

Bilirubin Uridine Diphospho-glucuronyltransferase in Rat Liver Microsomes: Genetic Variation and Maturation

Extract: Optimal conditions for the in vitro assay of bilirubin uridine diphospho- (UDP) glu-curonyltransferase activity in rat liver microsomes are described. Solvent partitioning was used to separate the conjugated from nonconjugated bilirubin, thus avoiding dependency on the rate of coupling with diazotized sulfanilic acid for the distinction between bilirubin and its conjugated form. The inclusion of uridine diphospho-N-ace-tylglucosamine (UDPNAG) in the reaction mixture permitted the rate of conjugation of bilirubin by fresh rat liver homogenates and microsomes to occur at greater saturation of the available enzyme with the substrates bilirubin and UDP-glucuronic acid. Liver microsomes, isolated in 0.15 M KC1, increased their activity for bilirubin conjugation and decreased their dependency on UDPNAG during the first 10 days of storage at — 15°. Chromatographic separation of the azo pigments of the conjugated bilirubin gave evidence to suggest that bilirubin monoglucuronide was the initial product and bilirubin diglucuronide appeared in increasing amounts in more prolonged incubations. These results suggested that bilirubin monoglucuronide can be intermediate to the formation of bilirubin diglucuronide. Bilirubin UDP-glucuronyltrans-ferase activity in hepatic microsomes of adult homozygous Gunn rats was not demonstrable. In microsomes of heterozygous Gunn rats and normal Wistar and Sprague-Dawley rats bilirubin UDP-glucuronyltransferase activity was found to be 31.0 and 58.0 μg bilirubin conjugated/mg microsomal N/30 min, respectively. Measurements in developing rats indicated that the maturation in enzyme activity occurred by at least two distinct means: increase of specific activity of the microsomes, and an increase in the content of microsomes per gram of liver (Table IV).Speculation: A method for quantitative measurement of bilirubin UDP-glucuronyltransferase activity applicable to samples obtained by needle biopsy has been needed. The method described in this report meets this need and may permit more precise differential diagnosis of retention jaundice of infancy and childhood and especially the “physiologic” jaundice of the newborn.

The photodynamic action of bilirubin on erythrocytes

Erythrocyte suspensions exposed to bilirubin in concentrations that frequently occur during neonatal life are hemolyzed when irradiated with fluorescent light. The hemolysis is preceded by membrane damage that is reflected by a loss of erythrocyte potassium and a reduction in membrane ATPase activity. The initiation of the cation loss requires the simultaneous presence of molecular oxygen and light and therefore involves a photodynamic action of bilirubin. The development of anemia associated with phototherapy may be a consequence of an in vivo photosensitized hemolysis.

Physiologic Hyperbilirubinemia in the Neonatal Period

THE degree and duration of hyperbilirubinemia during neonatal life result from many influences unique to the newborn period that serve either to increase the rate of formation of bilirubin or to re...

Protection from Bilirubin Nephropathy in Jaundiced Gunn Rats

The inclusion of 2% agar (w/w) in the diet of homozygous jaundiced (jj) rats resulted in greater fecal excretions of bile pigment, as measured by bilirubin and urobilin. Associated with the agar diet feedings, the concentrations of bilirubin in serum, adipose tissue, and renal medullae were less than in littermate animals fed the control diet. The renal free water clearance ( C H 2 O ) in jj animals was significantly lower by 5 months of age when compared with nonjaundiced (Jj) normal animals. The C H 2 O in jj animals raised on the agar diet did not significantly differ from normal Jj animals at 5 months of age and was greater than the littermate jj animals raised on the standard diet. The results suggest that the nephropathy of jj rats is secondary to the accumulation of bilirubin in the renal papillae rather than a genetic deficiency of medullary function.