Bilirubin is formed from breakdown of hemoglobin molecules by the reticuloendothelial system. Newly formed (unconjugated) bilirubin circulates in blood bound nonpermanently to serum albumin and is carried to the liver, where it is extracted by hepatic parenchymal cells, conjugated first with one and then with a second glucuronide molecule to form bilirubin diglucuronide, and then excreted in the bile. The bile passes through the common bile duct into the duodenal segment of the small intestine. It has been well documented that when a certain diazo compound discovered by van den Bergh is added to conjugated bilirubin, there will be color development maximal within 1 minute. This fast-reacting bilirubin fraction consists of bilirubin monoglucoronide, bilirubin diglucoronide, and a third fraction consisting of conjugated bilirubin (monoglucoronide) bound permanently and covalently to albumin, called “delta bilirubin.” If alcohol is then added, additional color development takes place for up to 30 minutes. This second component, which precipitates with alcohol, corresponds to unconjugated bilirubin. Actually color continues to develop slowly up to 15 minutes after the simple van den Bergh reaction maximal at 1 minute, and this extra fraction used to be known as the “delayed,” or “biphasic reaction.” It has since been shown that a considerable proportion of the substance involved is really unconjugated bilirubin. Since unconjugated bilirubin is measured more completely by the 30-minute alcohol precipitation technique, most laboratories do not report the biphasic reaction. The 1-minute van den Bergh color reaction is also called the “direct reaction” and the conjugated bilirubin it measures is known as “direct-acting bilirubin,” whereas the 30-minute alcohol measurement of unconjugated bilirubin is called the “indirect reaction” and its substrate is “indirect bilirubin.” Reference values are less than 1.5 mg/100 ml (25 µmol/L) for total bilirubin and less than 0.4 mg/100 ml (range, 0.2-0.5 mg/100 ml [3.42-8.55 µmol/L] depending on the method used) for 1-minute (“direct”) bilirubin.

There are certain problems with current measurements of conjugated bilirubin that affect clinical interpretation, especially regarding neonatal bilirubin. For many years the terms “direct” and “conjugated” bilirubin were used interchangeably, and in fact, were not far from the same using standard manual methods. However, with bilirubin now being assayed predominately by automated chemistry equipment, it is becoming evident that various combinations of reagents and automated equipment vary significantly in how much unconjugated bilirubin and delta bilirubin are included in “conjugated” bilirubin assay results. Therefore, it might be more realistic to use the old terminology (direct bilirubin). For example, in one specimen from a national organization’s proficiency test program that was supposed to contain 0.3 mg/100 ml (5.13 µmol/L) of conjugated bilirubin, six different instrument/reagent combinations obtained a value of 0.26 mg/100 ml (4.4 µmol/L) or less; three other instrument/reagent combinations reported a value of 1.04-1.13 mg/100 ml (17.7-19.3 µmol/L); and seven instrument/reagent combinations returned values between 0.26 and 1.04 mg/100 ml (4.4-17.8 µmol/L). All of these laboratories obtained approximately the same value for total bilirubin. Our hospital had one of the high-result chemistry instruments. We then measured conjugated (direct) bilirubin in 65 newborn infants who had jaundice and total bilirubin levels between 10.0 and 25.5 mg/100 ml (171-436.1 µmol/L). In these clinically healthy newborns, nearly all of the total bilirubin would be expected to be unconjugated. We obtained a reference range of 0.1-0.4 mg/100 ml (1.71-6.84 µmol/L) in clinically normal adults. In the newborns, we obtained direct bilirubin values ranging from 0.5-1.6 mg/100 ml (8.55-27.4 µmol/L), with 26% of cases being between 1.1-1.6 mg/100 ml (18.8-27.4 µmol/L), approximately 3-4 times the upper normal limits. This would produce difficulty in interpreting infant bilirubin levels because conditions such as sepsis, hepatitis due to different viruses, medication effects on the liver, biliary atresia, galactosemia, congenital bilirubin conjugation defects, and alpha-1 antitrypsin deficiency traditionally elevate conjugated bilirubin values. Interestingly, these falsely elevated values had a somewhat random distribution within the otherwise stepwise increase following the increase in total bilirubin, and there appeared to be a limit to the false increase. Therefore, a considerable number of automated instruments are falsely reporting a certain unpredictable amount of unconjugated bilirubin as direct or conjugated bilirubin and are not adjusting their direct bilirubin reference range to take this into account.

Another problem is that delta bilirubin may sometimes be included to some extent in direct bilirubin assay. Since delta bilirubin is bound tightly to serum albumin (which has a half-life of 19 days), this would cause apparent persistence of elevated serum direct bilirubin values (and therefore, comparable increase in total bilirubin) for several days after actual conjugated bilirubin levels had begun to fall.

Conjugated bilirubin in serum is excreted in urine until the renal threshold of 29 mg/100 ml (495 µmol/L) is exceeded. Although there is no exact correlation, a general trend has been reported toward a rising serum conjugated bilirubin level as the serum creatinine level rises (if the serum creatinine changes are due to renal disease). Since conjugated bilirubin is also excreted into the intestine through the biliary duct system, the influence of renal function would only be evident if abnormal quantities of conjugated bilirubin were present in patient serum.

Fasting, especially if prolonged, can increase total bilirubin values with normal proportions of conjugated and nonconjugated fractions. In one study, overnight fasting increased total bilirubin by an average of about 0.5 mg/100 ml (8.55 µmol/L) and 0.1-1.3 mg/100 ml (1.71-22.2 µmol/L). Poor renal function may decrease or delay excretion of conjugated bilirubin.

Visible bile staining of tissue is called “jaundice.” Three major causes predominate: hemolysis, extrahepatic biliary tract obstruction, and intrahepatic biliary tract obstruction.

Hemolysis causes increased breakdown of red blood cells (RBCs) and thus increased formation of unconjugated bilirubin. If hemolysis is severe enough, more unconjugated bilirubin may be present in the plasma than the liver can extract. Therefore, the level of total bilirubin will rise, with most of the rise due to the unconjugated fraction. The conjugated fraction remains normal or is only slightly elevated and rarely becomes greater than 1.2 mg/100 ml (20 µmol/L; unless a nonhemolytic problem is superimposed). Hemolysis may result from congenital hemolytic anemia (e.g., sickle cell anemia or other hemoglobinopathies), drug-induced causes, autoimmune disease, and transfusion reactions. An increased unconjugated bilirubin level may sometimes result from absorption of hemoglobin from extravascular hematomas or from pulmonary infarction. The unconjugated bilirubin level may also be increased in various other conditions mg/100 ml (20 µmol/L; unless a nonhemolytic problem is superimposed). Hemolysis may result from congenital hemolytic anemia (e.g., sickle cell anemia or other hemoglobinopathies), drug-induced causes, autoimmune disease, and transfusion reactions. An increased unconjugated bilirubin level may sometimes result from absorption of hemoglobin from extravascular hematomas or from pulmonary infarction. The unconjugated bilirubin level may also be increased in various other conditions. The reason is sometimes obscure. In most patients with most conditions producing unconjugated hyperbilirubinemia, the unconjugated fraction is usually less than 6 mg/100 ml (102.6 µmol/L) except for the rare Arias syndrome. In “pure” unconjugated hyperbilirubinemia, the unconjugated fraction is over 80% of total bilirubin. In one study of patients with unconjugated bilirubinemia, the most common associated diseases (collectively 60% of the total cases) included cholecystitis, cardiac disease (only 50% having overt congestive failure), acute or chronic infection, gastrointestinal (GI) tract disease (mostly ulcerative or inflammatory), and cancer.

Extrahepatic biliary tract obstruction is caused by common bile duct obstruction, usually due to a stone or to carcinoma from the head of the pancreas. The height of the total serum bilirubin level depends on whether the obstruction is complete or only partial and how long the obstruction has existed. Also, in the majority of cases the bilirubin level stabilizes at less than 20 mg/100 ml even when there is complete common bile duct obstruction. Extrahepatic biliary tract obstruction initially produces an increase in conjugated bilirubin without affecting the unconjugated bilirubin, since obstruction of the common bile duct prevents excretion of already conjugated bilirubin into the duodenum. However, after several days, some of the conjugated bilirubin in the blood breaks down to unconjugated bilirubin. Eventually the ratio of conjugated to unconjugated bilirubin approaches 1:1. The amount of time necessary for this change in the composition of the serum bilirubin is quite variable, but there is some correlation with the amount of time that has elapsed since onset of obstruction. In addition, prolonged intrahepatic bile stasis (“cholestasis”) due to extrahepatic obstruction of the bile drainage system often produces some degree of secondary liver cell damage, which also helps to change the ratio of conjugated to unconjugated bilirubin.

Unconjugated Hyperbilirubinemia
A. Due to increased bilirubin production (if normal liver, serum unconjugated bilirubin is usually less than 4 mg/100 ml)
1. Hemolytic anemia
a) Acquired
b) Congenital
2. Resorption from extravascular sources
a) Hematomas
b) Pulmonary infarcts
3. Excessive ineffective erythropoiesis
a) Congenital (congenital dyserythropoietic anemias)
b) Acquired (pernicious anemia, severe lead poisoning; if present, bilirubinemia is usually mild)
B. Defective hepatic unconjugated bilirubin clearance (defective uptake or conjugation)
1. Severe liver disease
2. Gilbert’s syndrome
3. Crigler-Najjar type I or II
4. Drug-induced inhibition
5. Portacaval shunt
6. Congestive heart failure
7. Hyperthyroidism (uncommon)

Intrahepatic biliary tract obstruction is usually caused by liver cell injury. The injured cells may obstruct small biliary channels between liver cell groups. Some bilirubin may be released from damaged cells. Liver cell injury may be produced by a wide variety of etiologies, such as alcohol- or drug-induced liver injury; acute or chronic hepatitis virus hepatitis; certain other viruses such as Epstein-Barr (infectious mononucleosis) or cytomegalovirus; active cirrhosis; liver passive congestion, primary or metastatic liver tumor; severe bacterial infection; and biliary cirrhosis. When serum bilirubin is increased due to liver cell damage, both conjugated and unconjugated bilirubin fractions may increase in varying proportions. The unconjugated fraction may be increased because of inability of the damaged cells to conjugate normal amounts of unconjugated serum bilirubin. The conjugated fraction increase usually results from intrahepatic cholestasis secondary to bile sinusoid blockage by damaged hepatic cells.

Other etiologies of jaundice. Carcinoma may increase serum bilirubin; either predominantly conjugated hyperbilirubin (if the common bile duct is obstructed) or by a variable mixture of conjugated and unconjugated bilirubin (if the tumor is intrahepatic). Intrahepatic tumor can obstruct intrahepatic bile ducts or destroy liver cells by compression from expanding tumor masses or by invasion and replacement of liver tissue. Total bilirubin is increased in about 45% of patients with liver metastases, with the incidence of hyperbilirubinemia reported to be about 70% for metastatic biliary tract and pancreatic carcinoma, about 50% for breast and lung carcinoma, about 35% for colon and gastric carcinoma, and about 10% for other tumors. In pancreatic, colon, and gastric metastatic tumor, total bilirubin may exceed 10 mg/100 ml in 10% or more patients. Drugs may produce bilirubin elevation of variable type and degree. Some drugs exert predominantly cholestatic (obstructive) effects, others induce hepatocellular injury, and still others have components of both cholestasis and hepatic cell injury. There are a large number of conditions that may produce elevated serum bilirubin levels with or without visible jaundice. Septicemia is one cause that is often not mentioned but that should not be forgotten. One report noted that in patients less than age 30 years, viral infections of the liver accounted for 80% of cases with jaundice. In the age group 30-60 years, viral infections accounted for 30%; alcoholic liver disease accounted for 30%, and gallstones or cancer accounted for about 10% each. Over age 60, cancer accounted for 45% of cases; gallstones accounted for 25%, and alcoholic liver disease and medications accounted for about 10% each.