بیلیروبین تک جفتی و درصد رو به افزون تک جفت‌های بیلیروبین در زرداب بیماران مبتلا به علائم Gilbert و بیماری Crigler- Najjar Unconjugated Bilirubin and an Increased Proportion of Bilirubin Monoconjugates in the Bile of Patients with Gilbert’s Syndrome and Crigler-Najjar Disease

Gilbert’s syndrome or “constitutional hepatic dysfunction,” is characterized by chronic, often mild, unconjugated hyperbilirubinemia in the absence of overt hemolysis (for a review, see reference 46). It has long remained a diagnosis made by exclusion of other diseases, and therefore depended on the extent of the investigations that had been performed. The recent (lemonstration of markedly decreased bilirubin UDPGTA, both in unactivated (5) and in digitonin-activated liver homogenates (2-4, 6, 7), complemented the previous observations of decreased transferase activity in some patients with more pronounced unconjugated hyperbilirubinemia (47, 48). In 1969, Arias et al. (22) described 16 cases of severe chronic nonhemolytic unconjugated hyperbilirubinemia which they proposed to subdivide into two groups. Group I was composed of the most severely affected children who developed kernicterus and whose condition did not improve upon treatment with phenobarbital. Their bile was “virtually colorless and contained only a trace of unconjugated bilirubin” (cited from Arias et al. [22]). In contrast, conjugates were detected in the bile of patients in group II, and their bilirubinemia decreased on treatment with phenobarbital. The activity of hepatic bilirubin UDP-GTA was near to zero in both groups. Further aspects of the disease have been discussed recently by Blaschke et al. (49). In the present study, several parameters of bilirubin metabolismii have been investigated in both disorders. In our patients with Gilbert’s syndrome, transferase activity was 4-45% of the control values. Zero or near-zero activities were found in the children with Crigler-Najjar disease, in agreement with other work (22, 49, 50). This, however, does not offer an absolute diagnostic criterion, as values approaching zero were occasionally found in patients with Gilbert’s syndrome (50, 51) and in neonates without liver disease (52). However, for the present, transferase assays allow differentiation of Gilbert’s syndrome and Crigler-Najjar disease from other types of unconjugated hyperbilirubinemia. The analysis of bile showed that conjugated bilirubin-IXa was present in all samples examined, including those obtained from the children with CriglerNajjar disease. The conjugating groups detected were glucuronic acid, glucose, and xylose. In seven of nine children with Crigler-Najjar disease, 9-48% of the EAazopigments contained glucuronic acid (8-azopigment); in two children (E and V), only trace amounts were found (Table I). Glucose residues (3-17%) were usually present in slightly higher relative proportions than in the bile from the controls. The present results indicate that the detectability or nondetectability of conjugated bilirubin-IXa (22) is not a reliable criterion for the differentiation of patients with Crigler-Najjar disease, although it should be noted that children E and V, whose bile contained only traces of glucuronide, belonged to the more severely affected group. The usefulness of differentiation based on response to phenobarbital (group II) or lack of response and development of kernicterus (group I) was confirmed (22). However, colorless bile was never observed in any of our children with Crigler-Najjar disease, even in the most severe cases. The most striking feature of the present study was the highly significant increase in azopigment a0 (unconjugated azodipyrrole) in the EA (pH 2.7)-treated bile from all patients with congenital nonhemolytic unconjugated hyperbilirubinemia. Minor increases in this pigment have occasionally been detected in patients with liver disease and cholestasis, but were accomnpanied by other specific chaniges in biliary bilirubini compositioni (23). The a0-fraction was significanitly higher in Crigler-Najjar disease than in Gilbert’s syndlromiie. A similar increase has been recently coInfirmned in four adults with Crigler-Naijar (lisease type II (53, 54). As the a0-fraction was not increase(d in patients with hemolysis in the absence of associated Gilbert’s syndrome (Table IV), determination of the a0- fraction imay allow an easy, safe, and rapid diagnosis of bilirubin-IXa UDP-GTA deficiency (Fig. 2). In patients with Gilbert’s syndrome, the deficiency is clearly expressed in decreased formation and biliary secretion of dliconijugated bilirubin-IXa In patients with Crigler-Najjar disease, our maini findinig was that conjugates were always present to some extent in bile, whatever the severity of the disease. Evidence was found that these conjugates were mainly, and perhaps exclusively, bilirubin-IXa monioglucuroniide, an observation which is in accordanice with a study by Gordon et al. (54), who showed in addition that there was a definite decrease in the total biliary bilirubin output in these patients. This clearly indicates that the maini abnormality in Crigler-Najjar disease is the decreased excretion of dliconijugates, reflecting the transferase deficienicy which is miiore pronounced in this condition than in Gilbert’s syndrome. In addition, the percentage of unlconijugated bilirubin-IXa that we found in bile was typically increased to 30-57% of the total bile pigment. Such an increase has also been found by isotopical mlethods (55, 56). However, the concentrations of unconjugated bilirubin-IXa in the bile of patients with Crigler-Najjar disease were only one- to sevenfold the values foundl in normal adults oir patients with Gilbert’s syndrome. This pigment therefore constitutes only a minor fraction of total heme breakdown. A comparable situation is found in Gunn rat bile where 31-40% of the biliary bile pigments is unconjugated bilirubinIXa, although the daily excretion of this unconjugated pigment is barely higher than that of control Wistar rats and approaches only 3-4% of total heme turnover (43). The observations reported in the present work raise several problems regarding the biochemical defin ition of the enzyme deficiencies involved in congenital, nonhemolytic, unconjugated hyperbilirubinemia. Firstly, a disproportion is apparent between transferase activities and the rates of formation of conjugated bilirubin-IXa in vivo. The near-zero GTA levels found occasionally in the liver of Gilbert’s patients in previous studies (50, 51), in the present work, and in neonatal liver (52), suggest that the standard enzyme assay which we have employed and which is optimized for normal liver tissue, may not always be adequate. The simplest explanation would appear to be that in Gilbert’s syndrome anl abniormiial transferase is present in the liver. A situation of this sort seems to exist in Gunn rats whose liver has a normal amounit of UDP-GTA actinig on p-nitrophenol with an abnormally low affinity for UDP-glucuronic acid (57). The near-zero tranisferase activities typically found in the liver of CriglerNajjar patients (Table 1) (22, 49, 50) are consistent with the low biliary output of bilirubin con-jugates in this condition (54, 55). The difference in response towards phenobarbital found in type I and type II patients may also point to a fundamental differenice in the type of transferase responsible for conjugatinig bilirubin-IXa. How can the decreased excretion of disconjugated bilirubin-IXa be related to the dlocumented bilirubinIXa UDP-GTA deficiency? Too little is known about the biochemical mechanisms underlying the conjugation and excretion of bilirubin to warrant anv thorough discussion. It may suffice to conisider briefly a few of many possibilities which couild explain our basic observations. If bilirubin-IXa were converted to its monoglucuronide and subsequently to its diglucuronide by a single enzymic site, then transferase deficiency would of course result in an increased bilirubin concentration in the cytosol. If one now assunmes that unconjugated bilirubin-IXa binds imiore strongly to the enzyme than its monocon-jugate, then a smnaller fraction of the enzymic sites would be free to bind the monoglucuronide under conditions when a decreased amount of enzyme is available. As a consequence, a less rapid conversion to the diconjugate would ensue, together perhaps with a more efficient direct biliary elimination of the monoglucuro nide. In the case of a two-enzyme system, an associated deficiency of the enzyme catalyzing conversion of mono- to diconjugated bilirubin-IXa could explain the present data. However, even if the activity of the second enzyme is normal in vitro, as proposed by Jansen et al. (58) in studies of two patients with Crigler-Najiar type IL, other mechaniism,is, such as accumulation of bilirubin-IXa within the hepatocyte, may inhibit the second step in vivo. Clearly, further investigation is necessary to elucidate the underlying mechanisms of glucuronide formiation.