The Synthesis of Bilirubin Glucuronide in Animal and Human Liver

Lathe and Walker (1957) investigated the conjugation of bilirubin with glucuronic acid. Although bilirubin is excreted as a glucuronide it was not known whether the system conjugating various alcohols and acids with glucuronic acid in the liver was responsible for the removal of bilirubin also. This detoxification system is located in liver microsomes and requires uridine diphosphate glucuronic acid (UDP glucuronic acid). In addition, the authors hoped to be able to shed light on the reduced capacity of new-born infants to excrete bilirubin.

Following materials and methods were used: The enzyme required to catalyze the conjugation was b-glucuronidase, an ox-liver preparation, supplied by a commercial laboratory. Uridine diphosphate glucuronic acid was obtained from liver slices of the various animals investigated. Bile was collected from the animals through the canulated bile duct. A solution consisting of b-glucuronidase, glucuronic acid, bilirubin and serum was incubated. The conjugated bilirubin in the incubation media was estimated by a direct diazo reaction before and after the incubation. After incubation the solution was centrifuged and the supernatant was read in a spectrophotometer at 525 mm. The results from the various tests were compared with a control preparation without th diazo solution and the difference indicated the amount of conjugated pigment.

Various animals were investigated. It was found that after the intravenous injection of bilirubin into a rat the concentration of both bilirubin and glucuronic acid in the bile rose. Rat-liver slices incubated in phosphate-bicarbonate solution containing bilirubin without serum conjugated the pigment very slowly. Addition of serum increased the rate. In several experiments the pH of the mixture was varied. It was found that conjugation was greatest at a final pH of 6.6.

Livers of three premature human infants were also examined for their capacity to conjugate bilirubin. The rate of conjugation was significantly below those of the animals investigated. (8 mg/g of wet liver/hr for premature infants compared to 400 for a wistar rat). Comparing excretion of glucuronic acid by infants of less than 5.5 lbs birth weight was not significantly different from those with a birth weight greater than 6.5 lbs.

In their discussion, the authors stated that the appearance of increased amounts of glucuronic acid after the injection and the hydrolysis of the azo pigment by b-glucuronidase suggested that the rat excretes bilirubin as a glucuronide. In comparing their results with those of others, they felt that the most striking difference in experimental conditions was the necessity for including plasma protein in the medium for conjugating bilirubin. They believed that the addition of serum provided substantial amounts of bilirubin in the medium without reducing the oxygen uptake of the cells.

Lathe GH and Walker M. The synthesis of bilirubin glucuronide in animal and human liver. Biochem J. 1958. 705-712.