ExtractOptimal conditions for the in vitro assay of bilirubin uridine diphospho-(UDP) glucuronyltransferase 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-iV-acetylglucosamine (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-glucuronyltransferase activity in hepatic microsomes of adult homozygous Gunn rats was not demonstrable. In microsomes of heterozygous Gunn rats and normal Wistar and SpragueDawley rats bilirubin UDP-glucuronyltransferase activity was found to be 31.0 and 58.0 yug 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).
The controlled clinical observations of Silverman and coworkers 1 presented the first evidence that drugs bound to albumin could be associated with an increased incidence of bilirubin toxicity. A three-fold increase in the incidence of bilirubin encephalopathy was found at autopsy in premature infants treated prophylactically with sulfisoxazole, and the treated infants had lower serum bilirubins than the control population. Further confirmation was provided by Johnson and associatesZ in jaundiced Gunn rats. They documented an abrupt decrease in the serum bilirubin concentration after injection of anions known to be bound by albumin and found a simultaneous increase in the incidence of bilirubin encephalopathy. By in vitro studies, I reported that addition of protein-bound anions to sera from jaundiced infants was associated with a decrease in the spectral absorption curve of protein-bound bilirubin, and after addition of these anions, some of the serum bilirubin was ultrafilterable. It was therefore proposed 4 that a critical determinant in the toxicity of bilirubin is the concentration of diffusible bilirubin rather than its total extracellular concentration. The bilirubin of interstitial fluid is represented, in FIGURE 1, as a stoichiometric equilibrium between an albumin-bilirubin ligand and unbound bilirubin. It is this latter fraction that, because of its lipid solubility, can readily penetrate plasma cell membranes and exert its toxicity. The presence of anions in the circulation that share a common binding site on albumin with bilirubin or indirectly reduce the affinity of albumin for bilirubin can enhance its cytotoxicity by increasing the concentration of diffusible bilirubin. Measurements of the relative saturation of albumin with bilirubin have permitted the detection of the infant at risk to bilirubin en~ephalopathy.~ These measurements can also provide a means to test drugs that are bound to albumin and thereby might increase the toxicity of bilirubin if used therapeutically in jaundiced newborn infants. The basis for the method we adopted is illustrated in FIGURE 2. Bilirubin, when dissolved in a protein-free solution, has an extinction maximum at 440 nm. An identical bilirubin solution that contains an equimolar amount of albumin exhibits a similar millimolar extinction coefficient, but the maximum is at 460 nm. There is a 14% difference in absorbance at 460 nm between free and protein-bound bilirubin, and therefore addition of a test anion that displaces bilirubin from albumin would be reflected by a decreased absorbance at 460 nm (AOD.,,, ,,,,). By calculating the percentage decrease in absorbance at 460 nm in comparison to an equally diluted control sample, the amount of displacement can be quantitated when corrected for nonbilirubin chromogens. The degree of displacement could theoretically vary between 0 and 100% and would be reflected by a percentage
ExtractLitter-mate homozygous and heterozygous weanling rats of the Gunn strain were thirsted and fasted for 24 hours. The response of the animals to thirsting was compared by analysis of solute content of serum and composition of fluid in the renal medullae. The results are given in tables I and II. When the mean values for weight loss and serum solute concentrations were compared, it was found that the jaundiced rats were able to conserve body water as well as the non-jaundiced rats and to generate equally high concentrations of total solute in the tissue water of the renal medulla. Only two of the twelve jaundiced animals showed evidence of failure to develop the expected hypertonicity of the medullary tissue water. This failure was qualitatively similar to that found in adult jaundiced rats. Despite the presence of higher concentrations of bilirubin in serum, the medullary bilirubin content of the jaundiced weanling rat was only 5 % of the concentration found in adult animals. The sodium and urea concentrations in the renal medullary fluid were correlated (p <0.001) in the homozygous jaundiced rats (table IV and fig. 1). A similar correlation was not found in the non-jaundiced heterozygous animals. This finding suggests that the normal rat kidney has at least two mechanisms for renal transport of urea, one of which is sodium dependent and the other sodium independent and that the latter mechanism is inoperative in the jaundiced weanling rat.
SpeculationIn vitro studies of bilirubin have previously suggested that toxicity is exerted by its surface activity on mitochondrial membranes. The examination of renal function in the intact jaundiced animal has revealed the possibility that tubular transport systems for urea and sodium are impaired in the presence of high concentrations of bilirubin in the renal medulla. These transport systems involve the maintenance of concentration gradients of sodium and urea across tubular epithelium. Bilirubin, through its surface activity, could impair the selective permeability of the membranes involved in urea and sodium transport within the renal medulla.
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