INCREASED BILIRUBIN PRODUCTION
Increased destruction of erythrocytes leads to increased bilirubin turnover and unconjugated hyperbilirubinemia; the hyperbilirubinemia is usually modest in the presence of normal liver function. In particular, the bone marrow is only capable of a sustained eightfold increase in erythrocyte production in response to a hemolytic stress. Therefore, hemolysis alone cannot result in a sustained hyperbilirubinemia of more than ~68 μmol/L (4 mg/dL). Higher values imply concomitant hepatic dysfunction. When hemolysis is the only abnormality in an otherwise healthy individual, the result is a purely unconjugated hyperbilirubinemia, with the direct-reacting fraction as measured in a typical clinical laboratory being ≤15% of the total serum bilirubin. In the presence of systemic disease, which may include a degree of hepatic dysfunction, hemolysis may produce a component of conjugated hyperbilirubinemia in addition to an elevated unconjugated bilirubin concentration. Prolonged hemolysis may lead to the precipitation of bilirubin salts within the gallbladder or biliary tree, resulting in the formation of gallstones in which bilirubin, rather than cholesterol, is the major component. Such pigment stones may lead to acute or chronic cholecystitis, biliary obstruction, or any other biliary tract consequence of calculous disease.
During erythroid maturation, small amounts of hemoglobin may be lost at the time of nuclear extrusion, and a fraction of developing erythroid cells is destroyed within the marrow. These processes normally account for a small proportion of bilirubin that is produced. In various disorders, including thalassemia major, megaloblastic anemias due to folate or vitamin B12 deficiency, congenital erythropoietic porphyria, lead poisoning, and various congenital and acquired dyserythropoietic anemias, the fraction of total bilirubin production derived from ineffective erythropoiesis is increased, reaching as much as 70% of the total. This may be sufficient to produce modest degrees of unconjugated hyperbilirubinemia.
Degradation of the hemoglobin of extravascular collections of erythrocytes, such as those seen in massive tissue infarctions or large hematomas, may lead transiently to unconjugated hyperbilirubinemia.
DECREASED HEPATIC BILIRUBIN CLEARANCE
Decreased hepatic bilirubin uptake is believed to contribute to the unconjugated hyperbilirubinemia of Gilbert’s syndrome (GS), although the molecular basis for this finding remains unclear (see below). Several drugs, including flavaspidic acid, novobiocin, and rifampin, as well as various cholecystographic contrast agents, have been reported to inhibit bilirubin uptake. The resulting unconjugated hyperbilirubinemia resolves with cessation of the medication.
PHYSIOLOGIC NEONATAL JAUNDICE
Bilirubin produced by the fetus is cleared by the placenta and eliminated by the maternal liver. Immediately after birth, the neonatal liver must assume responsibility for bilirubin clearance and excretion. However, many hepatic physiologic processes are incompletely developed at birth. Levels of UGT1A1 are low, and alternative excretory pathways allow passage of unconjugated bilirubin into the gut. Since the intestinal flora that convert bilirubin to urobilinogen are also undeveloped, an enterohepatic circulation of unconjugated bilirubin ensues. As a consequence, most neonates develop mild unconjugated hyperbilirubinemia between days 2 and 5 after birth. Peak levels are typically <85–170 μmol/L (5–10 mg/dL) and decline to normal adult concentrations within 2 weeks, as mechanisms required for bilirubin disposition mature. Prematurity, often associated with more profound immaturity of hepatic function and hemolysis, can result in higher levels of unconjugated hyperbilirubinemia. A rapidly rising unconjugated bilirubin concentration, or absolute levels >340 μmol/L (20 mg/dL), puts the infant at risk for bilirubin encephalopathy, or kernicterus. Under these circumstances, bilirubin crosses an immature blood-brain barrier and precipitates in the basal ganglia and other areas of the brain. The consequences range from appreciable neurologic deficits to death. Treatment options include phototherapy, which converts bilirubin into water-soluble photoisomers that are excreted directly into bile, and exchange transfusion. The canalicular mechanisms responsible for bilirubin excretion are also immature at birth, and their maturation may lag behind that of UGT1A1; this can lead to transient conjugated neonatal hyperbilirubinemia, especially in infants with hemolysis.
ACQUIRED CONJUGATION DEFECTS
A modest reduction in bilirubin conjugating capacity may be observed in advanced hepatitis or cirrhosis. However, in this setting, conjugation is better preserved than other aspects of bilirubin disposition, such as canalicular excretion. Various drugs, including pregnanediol, novobiocin, chloramphenicol, gentamicin, and atazanavir may produce unconjugated hyperbilirubinemia by inhibiting UGT1A1 activity. Bilirubin conjugation may be inhibited by certain fatty acids that are present in breast milk, but not serum of mothers whose infants have excessive neonatal hyperbilirubinemia (breast milk jaundice). Alternatively, there may be increased enterohepatic circulation of bilirubin in these infants. The pathogenesis of breast milk jaundice appears to differ from that of transient familial neonatal hyperbilirubinemia (Lucey-Driscoll syndrome), in which there may be a UGT1A1 inhibitor in maternal serum.
HEREDITARY DEFECTS IN BILIRUBIN CONJUGATION
Three familial disorders characterized by differing degrees of unconjugated hyperbilirubinemia have long been recognized. The defining clinical features of each are described below (Table 331-1). While these disorders have been recognized for decades to reflect differing degrees of deficiency in the ability to conjugate bilirubin, recent advances in the molecular biology of the UGT1 gene complex have elucidated their interrelationships and clarified previously puzzling features.
TABLE 331-1Principal Differential Characteristics of Gilbert’s and Crigler-Najjar Syndromes ||Download (.pdf) TABLE 331-1 Principal Differential Characteristics of Gilbert’s and Crigler-Najjar Syndromes
|Feature ||Crigler-Najjar Syndrome ||Gilbert’s Syndrome |
|Type I ||Type II |
|Total serum bilirubin, μmol/L (mg/dL) ||310–755 (usually >345) (18–45 [usually >20]) ||100–430 (usually ≤345) (6–25 [usually ≤20]) ||Typically ≤70 μmol/L (≤4 mg/dL) in absence of fasting or hemolysis |
Routine liver tests
Response to phenobarbital
Decreases bilirubin by >25%
Decreases bilirubin to normal
Usually normal; increased lipofuscin pigment in some
Pale or colorless
Largest fraction (mean: 57%) monoconjugates
Normal dark color
Mainly diconjugates but monoconjugates increased (mean: 23%)
Bilirubin UDP-glucuronosyltransferase activity
Inheritance (all autosomal)
Typically absent; traces in some patients
Markedly reduced: 0–10% of normal
Reduced: typically 10–33% of normal
Promoter mutation: recessive
Missense mutations: 7 of 8 dominant; 1 reportedly recessive
Crigler-Najjar Syndrome, Type I
CN-I is characterized by striking unconjugated hyperbilirubinemia of about 340–765 μmol/L (20–45 mg/dL) that appears in the neonatal period and persists for life. Other conventional hepatic biochemical tests such as serum aminotransferases and alkaline phosphatase are normal, and there is no evidence of hemolysis. Hepatic histology is also essentially normal except for the occasional presence of bile plugs within canaliculi. Bilirubin glucuronides are virtually absent from the bile, and there is no detectable constitutive expression of UGT1A1 activity in hepatic tissue. Neither UGT1A1 activity nor the serum bilirubin concentration responds to administration of phenobarbital or other enzyme inducers. Unconjugated bilirubin accumulates in plasma, from which it is eliminated very slowly by alternative pathways that include direct passage into the bile and small intestine, possibly via bilirubin photoisomers. This accounts for the small amount of urobilinogen found in feces. No bilirubin is found in the urine. First described in 1952, the disorder is rare (estimated prevalence, 0.6–1.0 per million). Many patients are from geographically or socially isolated communities in which consanguinity is common, and pedigree analyses show an autosomal recessive pattern of inheritance. The majority of patients (type IA) exhibit defects in the glucuronide conjugation of a spectrum of substrates in addition to bilirubin, including various drugs and other xenobiotics. These individuals have mutations in one of the common exons (2–5) of the UGT1 gene (Fig. 331-2). In a smaller subset (type IB), the defect is limited largely to bilirubin conjugation, and the causative mutation is in the bilirubin-specific exon A1. Estrogen glucuronidation is mediated by UGT1A1 and is defective in all CN-I patients. More than 30 different genetic lesions of UGT1A1 responsible for CN-I have been identified, including deletions, insertions, alterations in intron splice donor and acceptor sites, exon skipping, and point mutations that introduce premature stop codons or alter critical amino acids. Their common feature is that they all encode proteins with absent or, at most, traces of bilirubin-UDP-glucuronosyltransferase enzymatic activity.
Prior to the use of phototherapy, most patients with CN-I died of bilirubin encephalopathy (kernicterus) in infancy or early childhood. A few lived as long as early adult life without overt neurologic damage, although more subtle testing usually indicated mild but progressive brain damage. In the absence of liver transplantation, death eventually supervened from late-onset bilirubin encephalopathy, which often followed a nonspecific febrile illness. Although isolated hepatocyte transplantation has been used in a small number of cases of CN-I, early liver transplantation (Chap. 338) remains the best hope to prevent brain injury and death at present. It is anticipated that gene replacement therapy may be an option in the future.
Crigler-Najjar Syndrome, Type II (CN-II)
This condition was recognized as a distinct entity in 1962 and is characterized by marked unconjugated hyperbilirubinemia in the absence of abnormalities of other conventional hepatic biochemical tests, hepatic histology, or hemolysis. It differs from CN-I in several specific ways (Table 331-1): (1) Although there is considerable overlap, average bilirubin concentrations are lower in CN-II; (2) accordingly, CN-II is only infrequently associated with kernicterus; (3) bile is deeply colored, and bilirubin glucuronides are present, with a striking, characteristic increase in the proportion of monoglucuronides; (4) UGT1A1 in liver is usually present at reduced levels (typically ≤10% of normal); and (5) while typically detected in infancy, hyperbilirubinemia was not recognized in some cases until later in life and, in one instance, at age 34. As with CN-I, most CN-II cases exhibit abnormalities in the conjugation of other compounds, such as salicylamide and menthol, but in some instances, the defect appears limited to bilirubin. Reduction of serum bilirubin concentrations by >25% in response to enzyme inducers such as phenobarbital distinguishes CN-II from CN-I, although this response may not be elicited in early infancy and often is not accompanied by measurable UGT1A1 induction. Bilirubin concentrations during phenobarbital administration do not return to normal but are typically in the range of 51–86 μmol/L (3–5 mg/dL). Although the incidence of kernicterus in CN-II is low, instances have occurred, not only in infants but also in adolescents and adults, often in the setting of an intercurrent illness, fasting, or another factor that temporarily raises the serum bilirubin concentration above baseline and reduces serum albumin levels. For this reason, phenobarbital therapy is widely recommended, a single bedtime dose often sufficing to maintain clinically safe serum bilirubin concentrations.
Over 100 different mutations in the UGT1 gene have been identified as causing CN-I or CN-II. It was found that missense mutations are more common in CN-II patients, as would be expected in this less severe phenotype. Their common feature is that they encode for a bilirubin-UDP-glucuronosyltransferase with markedly reduced, but detectable, enzymatic activity. The spectrum of residual enzyme activity explains the spectrum of phenotypic severity of the resulting hyperbilirubinemia. Molecular analysis has established that a large majority of CN-II patients are either homozygotes or compound heterozygotes for CN-II mutations and that individuals carrying one mutated and one entirely normal allele have normal bilirubin concentrations.
This syndrome is characterized by mild unconjugated hyperbilirubinemia, normal values for standard hepatic biochemical tests, and normal hepatic histology other than a modest increase of lipofuscin pigment in some patients. Serum bilirubin concentrations are most often <51 μmol/L (<3 mg/dL), although both higher and lower values are frequent. The clinical spectrum of hyperbilirubinemia fades into that of CN-II at serum bilirubin concentrations of 86–136 μmol/L (5–8 mg/dL). At the other end of the scale, the distinction between mild cases of GS and a normal state is often blurred. Bilirubin concentrations may fluctuate substantially in any given individual, and at least 25% of patients will exhibit temporarily normal values during prolonged follow-up. More elevated values are associated with stress, fatigue, alcohol use, reduced caloric intake, and intercurrent illness, while increased caloric intake or administration of enzyme-inducing agents produces lower bilirubin levels. GS is most often diagnosed at or shortly after puberty or in adult life during routine examinations that include multichannel biochemical analyses. UGT1A1 activity is typically reduced to 10–35% of normal, and bile pigments exhibit a characteristic increase in bilirubin monoglucuronides. Studies of radiobilirubin kinetics indicate that hepatic bilirubin clearance is reduced to an average of one-third of normal. Administration of phenobarbital normalizes both the serum bilirubin concentration and hepatic bilirubin clearance; however, failure of UGT1A1 activity to improve in many such instances suggests the possible coexistence of an additional defect. Compartmental analysis of bilirubin kinetic data suggests that GS patients may have a defect in bilirubin uptake as well as in conjugation, although this has not been shown directly. Defect(s) in the hepatic uptake of other organic anions that at least partially share an uptake mechanism with bilirubin, such as sulfobromophthalein and indocyanine green (ICG), are observed in a minority of patients. The metabolism and transport of bile acids that do not utilize the bilirubin uptake mechanism are normal. The magnitude of changes in the serum bilirubin concentration induced by provocation tests such as 48 hours of fasting or the IV administration of nicotinic acid have been reported to be of help in separating GS patients from normal individuals. Other studies dispute this assertion. Moreover, on theoretical grounds, the results of such studies should provide no more information than simple measurements of the baseline serum bilirubin concentration. Family studies indicate that GS and hereditary hemolytic anemias such as hereditary spherocytosis, glucose-6-phosphate dehydrogenase deficiency, and β-thalassemia trait sort independently. Reports of hemolysis in up to 50% of GS patients are believed to reflect better case finding, since patients with both GS and hemolysis have higher bilirubin concentrations, and are more likely to be jaundiced, than patients with either defect alone.
GS is common, with many series placing its prevalence as high as 8%. Males predominate over females by reported ratios ranging from 1.5:1 to >7:1. However, these ratios may have a large artifactual component since normal males have higher mean bilirubin levels than normal females, but the diagnosis of GS is often based on comparison to normal ranges established in men. The high prevalence of GS in the general population may explain the reported frequency of mild unconjugated hyperbilirubinemia in liver transplant recipients. The disposition of most xenobiotics metabolized by glucuronidation appears to be normal in GS, as is oxidative drug metabolism in the majority of reported studies. The principal exception is the metabolism of the antitumor agent irinotecan (CPT-11), whose active metabolite (SN-38) is glucuronidated specifically by bilirubin-UDP-glucuronosyltransferase. Administration of CPT-11 to patients with GS has resulted in several toxicities, including intractable diarrhea and myelosuppression. Some reports also suggest abnormal disposition of menthol, estradiol benzoate, acetaminophen, tolbutamide, and rifamycin SV. Although some of these studies have been disputed, and there have been no reports of clinical complications from use of these agents in GS, prudence should be exercised in prescribing them, or any agents metabolized primarily by glucuronidation in this condition. It should also be noted that the HIV protease inhibitors indinavir and atazanavir (Chap. 197) can inhibit UGT1A1, resulting in hyperbilirubinemia that is most pronounced in patients with preexisting GS.
Most older pedigree studies of GS were consistent with autosomal dominant inheritance with variable expressivity. However, studies of the UGT1 gene in GS have indicated a variety of molecular genetic bases for the phenotypic picture and several different patterns of inheritance. Studies in Europe and the United States found that nearly all patients had normal coding regions for UGT1A1, but were homozygous for the insertion of an extra TA (i.e., A[TA]7TAA rather than A[TA]6TAA) in the promoter region of the first exon. This appeared to be necessary, but not sufficient, for clinically expressed GS, since 15% of normal controls were also homozygous for this variant. While normal by standard criteria, these individuals had somewhat higher bilirubin concentrations than the rest of the controls studied. Heterozygotes for this abnormality had bilirubin concentrations identical to those homozygous for the normal A[TA]6TAA allele. The prevalence of the A[TA]7TAA allele in a general Western population is 30%, in which case 9% would be homozygotes. This is slightly higher than the prevalence of GS based on purely phenotypic parameters. It was suggested that additional variables, such as mild hemolysis or a defect in bilirubin uptake, might be among the factors enhancing phenotypic expression of the defect.
Phenotypic expression of GS due solely to the A[TA]7TAA promoter abnormality is inherited as an autosomal recessive trait. A number of CN-II kindreds have been identified in whom there is also an allele containing a normal coding region but the A[TA]7TAA promoter abnormality. CN-II heterozygotes, who have the A[TA]6TAA promoter, are phenotypically normal, whereas those with the A[TA]7TAA promoter express the phenotypic picture of GS. GS in such kindreds may also result from homozygosity for the A[TA]7TAA promoter abnormality. Seven different missense mutations in the UGT1 gene that reportedly cause GS with dominant inheritance have been found in Japanese individuals. Another Japanese patient with mild unconjugated hyperbilirubinemia was homozygous for a missense mutation in exon 5. GS in her family appeared to be recessive.