Chowdhury J R, Chowdhury N R, Gärtner U, Wolkoff A W, Arias I M
J Clin Invest. 1982 Mar;69(3):595-603. doi: 10.1172/jci110486.
Bilirubin diglucuronide (BDG) may be formed in vitro by microsomal UDP glucuronosyl transferase (EC 2.4.1.17)-mediated transfer of a second mole of glucuronic acid from UDP-glucuronic acid, or by dismutation of bilirubin monoglucuronide (BMG) to BDG and unconjugated bilirubin, catalyzed by an enzyme (EC 2.4.1.95) that is concentrated in plasma membrane-enriched fractions of rat liver. To evaluate the role of these two enzymatic mechanisms in vivo, [(3)H]bilirubin mono-[(14)C]glucuronide was biosynthesized, purified by thin-layer chromatography, and tracer doses were infused intravenously in homozygous Gunn (UDP glucuronyl transferase-deficient) rats or Wistar rats. Bilirubin conjugates in bile were separated by high-pressure liquid chromatography and (3)H and (14)C were quantitated. In Gunn rats, the (14)C:(3)H ratio in BDG excreted in bile was twice the ratio in injected BMG. In Wistar rats the (14)C:(3)H ratio in biliary BDG was 1.25 +/- 0.06 (mean +/- SEM) times the ratio in injected BMG. When double labeled BMG was injected in Wistar rats after injection of excess unlabeled unconjugated bilirubin (1.7 mumol), the (14)C:(3)H ratio in BDG excreted in bile was identical to the ratio in injected BMG. Analysis of isomeric composition of bilirubin conjugates after alkaline hydrolysis or alkaline methanolysis indicated that the bile pigments retained the IX(alpha) configuration during these experiments. The results indicate that both enzymatic dismutation and UDP glucuronyl transferase function in vivo in BDG formation, and that dismutation is inhibited by a high intrahepatic concentration of unconjugated bilirubin. This hypothesis was supported by infusion of [(3)H]bilirubin-monoglucuronide in isolated perfused homozygous Gunn rat liver after depletion of intrahepatic bilirubin by perfusion with bovine serum albumin (2.5%), and after bilirubin repletion following perfusion with 0.34 mM bilirubin. From 20 to 25% of injected radioactivity was recovered in BDG in bile in the bilirubin-depleted state; only 8-10% of radioactivity was in BDG in bile after bilirubin repletion. After infusion of [(3)H]bilirubin di-[(14)C]glucuronide in homozygous Gunn rats, 5-7% of the injected pigment was excreted in bile as BMG. The (14)C:(3)H ratio in the injected BDG was 10% greater than the (14)C:(3)H ratio in BMG excreted in bile. These results indicate that in vivo, dismutation rather than partial hydrolysis, is responsible for BMG formation. Incubation of [(3)H]bilirubin, BDG and a rat liver plasma membrane preparation resulted in formation of BMG (3.3 nmol/min per mg protein) indicating that dismutation is also reversible in vitro.
胆红素双葡萄糖醛酸酯（BDG）可在体外通过微粒体UDP葡萄糖醛酸基转移酶（EC 2.4.1.17）介导的从UDP-葡萄糖醛酸转移第二摩尔葡萄糖醛酸而形成，或者通过胆红素单葡萄糖醛酸酯（BMG）歧化为BDG和未结合胆红素而形成，此反应由一种酶（EC 2.4.1.95）催化，该酶在大鼠肝脏富含质膜的组分中富集。为了评估这两种酶促机制在体内的作用，生物合成了[³H]胆红素单[¹⁴C]葡萄糖醛酸酯，通过薄层色谱法进行纯化，并将示踪剂量静脉注射到纯合Gunn（UDP葡萄糖醛酸基转移酶缺陷型）大鼠或Wistar大鼠体内。通过高压液相色谱法分离胆汁中的胆红素结合物，并对³H和¹⁴C进行定量。在Gunn大鼠中，胆汁中排泄的BDG中的¹⁴C:³H比值是注射的BMG中该比值的两倍。在Wistar大鼠中，胆汁中BDG的¹⁴C:³H比值是注射的BMG中该比值的1.25±0.06（平均值±标准误）倍。当在Wistar大鼠中注射过量未标记的未结合胆红素（1.7 μmol）后再注射双标记的BMG时，胆汁中排泄的BDG中的¹⁴C:³H比值与注射的BMG中的比值相同。对碱性水解或碱性甲醇解后胆红素结合物的异构体组成分析表明，在这些实验过程中胆色素保留了IX(α)构型。结果表明，酶促歧化和UDP葡萄糖醛酸基转移酶在体内BDG形成过程中均起作用，并且歧化受到肝内高浓度未结合胆红素的抑制。在用2.5%牛血清白蛋白灌注使肝内胆红素耗竭后，以及在用0.34 mM胆红素灌注使胆红素再充盈后，将[³H]胆红素单葡萄糖醛酸酯注入离体灌注的纯合Gunn大鼠肝脏，这一假设得到了支持。在胆红素耗竭状态下，胆汁中BDG中回收了20%至25%的注射放射性；胆红素再充盈后，胆汁中BDG中的放射性仅为8%至10%。在纯合Gunn大鼠中注射[³H]胆红素双[¹⁴C]葡萄糖醛酸酯后，5%至7%的注射色素以BMG形式排泄到胆汁中。注射的BDG中的¹⁴C:³H比值比胆汁中排泄的BMG中的¹⁴C:³H比值大10%。这些结果表明，在体内，是歧化而非部分水解导致BMG的形成。[³H]胆红素、BDG与大鼠肝脏质膜制剂一起孵育导致形成BMG（每毫克蛋白质3.3 nmol/分钟），表明歧化在体外也是可逆的。
