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Clinical aspects of serum bilirubin determination

Clinical aspects of serum bilirubin determination
Author:
Lawrence S Friedman, MD
Section Editor:
Sanjiv Chopra, MD, MACP
Deputy Editor:
Shilpa Grover, MD, MPH, AGAF
Literature review current through: Dec 2022. | This topic last updated: Sep 20, 2021.

INTRODUCTION — A number of blood tests are available that reflect the condition of the liver. The most common tests used in clinical practice include the serum aminotransferases, bilirubin, alkaline phosphatase, albumin, and prothrombin time. These tests are often referred to as "liver function tests," although this term is somewhat misleading since most do not accurately reflect how well the liver is functioning, and abnormal values can be caused by diseases unrelated to the liver. In addition, these tests may be normal in patients who have advanced liver disease.

Several specialized tests have also been developed (such as indocyanine green clearance), which, although uncommonly used in clinical practice, can measure specific aspects of hepatic function.

Despite their limitations, liver biochemical and function tests have many applications in clinical medicine:

They provide a noninvasive method to screen for the presence of liver disease. The serum alanine aminotransferase, for example, used to be part of panel of tests used to screen all blood donors in the United States for the presence of transmissible viruses.

They can be used to measure the efficacy of treatments for liver disease (such as immunosuppressant agents for autoimmune hepatitis). (See "Autoimmune hepatitis: Treatment".)

They can be used to monitor the progression of a disease such as viral or alcoholic hepatitis.

They can reflect the severity of liver disease, particularly in patients who have cirrhosis. As an example, the Child-Turcotte-Pugh score, which incorporates the prothrombin time and serum bilirubin and albumin concentrations, can predict survival (table 1).

The pattern of abnormalities on these tests is more accurate than any of the individual tests. Elevation of serum aminotransferases indicates hepatocellular injury, while elevation of the alkaline phosphatase indicates cholestasis. Recognition of patterns that are consistent with specific diseases can prompt appropriate additional testing.

The liver biochemical and function tests that are used commonly in clinical practice and that are used occasionally for specific circumstances can be categorized as follows:

Tests that detect injury to hepatocytes – Most of these tests measure the activity of hepatic enzymes, such as the aminotransferases, in the circulation. These enzymes are normally intracellular, but are released when hepatocytes are injured. (See "Liver biochemical tests that detect injury to hepatocytes".)

Tests of the liver's capacity to transport organic anions and metabolize drugs – These tests measure the liver's ability to clear endogenous or exogenous substances from the circulation. The best studied include serum measurements of bilirubin, bile acids, caffeine, and lidocaine metabolites, a variety of breath tests, and clearance tests such as bromsulphalein (BSP) and indocyanine green (ICG).

Tests of the liver's biosynthetic capacity – The most commonly performed tests to assess the biosynthetic capacity of the liver are the serum albumin and the prothrombin time (which requires the presence of clotting factors produced in the liver). Other tests that have been used are the serum concentrations of lipoproteins, ceruloplasmin, ferritin, and alpha 1-antitrypsin.

Tests that detect chronic inflammation in the liver, altered immunoregulation, or viral hepatitis – These tests include the immunoglobulins, hepatitis serologies, and specific autoantibodies. Most of these substances are proteins made by B lymphocytes, not by hepatocytes. However, some are quite specific for certain liver diseases, such as antimitochondrial antibodies in primary biliary cholangitis. (See "Clinical manifestations, diagnosis, and prognosis of primary biliary cholangitis (primary biliary cirrhosis)".)

This topic review will highlight practical considerations in serum bilirubin measurement. The approach to a patient who has hyperbilirubinemia is presented separately (see "Diagnostic approach to the adult with jaundice or asymptomatic hyperbilirubinemia"). Additional tests of the liver's capacity to transport organic anions and metabolize drugs as well as other categories of liver function tests are discussed separately. (See "Tests of the liver's capacity to transport organic anions and metabolize drugs".)

BILIRUBIN — Bilirubin is the catabolic product of heme metabolism, which is formed by the breakdown of heme present in hemoglobin, myoglobin, cytochromes, catalase, peroxidase, and tryptophan pyrrolase (figure 1). Eighty percent of the daily bilirubin production (250 to 400 mg) is derived from hemoglobin; the remaining 20 percent is contributed by other heme proteins and a small pool of free heme that turns over rapidly. Some of the bilirubin produced is then conjugated in the liver (figure 2) [1]. (See "Bilirubin metabolism".)

Mean total bilirubin levels are higher in males than in females and higher in White and Hispanic populations compared with Black populations. In the general population, serum bilirubin levels correlate with the risk of symptomatic gallstone disease [2] and inversely with the risk of stroke, respiratory disease, cardiovascular disease, peripheral artery disease, and mortality, presumably because bilirubin has antioxidant properties and perhaps because it is a signaling molecule [3]. Although, the serum bilirubin level has been reported to correlate with mortality in patients with acute myocardial infarction complicated by heart failure [2,4-9] and in patients with chronic obstructive lung disease [10].

Measurement of serum bilirubin — Several laboratory techniques have been developed for measuring the serum bilirubin concentration. The specific technique used has implications for the interpretation of serum values.

van den Bergh method — The terms "direct-" and "indirect-reacting" bilirubin were based upon laboratory techniques developed at the beginning of the 20th century that are still used in many clinical laboratories [11]. Direct and indirect bilirubin reflect the concentrations of conjugated and unconjugated bilirubin, respectively.

The technique involves the reaction of bilirubin with a diazo compound (diazotized sulfanilic acid), which creates two relatively stable dipyrryl azopigments. These fractions can be detected spectrophotometrically (their maximal absorption occurs at 540 nm). The indirect and direct fractions can be distinguished based upon their rate of production in the absence or presence of alcohol. The fraction produced within one minute in the absence of alcohol represents the concentration of direct bilirubin; the total serum bilirubin is that amount that reacts in 30 minutes after the addition of alcohol; and the indirect fraction is the difference between the total and the direct bilirubin. The fast reaction of direct (conjugated bilirubin) is due to the absence of internal hydrogen bonding and the fact that it is water soluble.

Total serum bilirubin concentrations using this technique are between 0.2 and 0.9 mg/dL (2 to 15.4 micromol/L) in 95 percent of the general population, and below 1 mg/dL (18 micromol/L) in 99 percent [11,12]. Direct bilirubin represents up to 30 percent, or 0.3 mg/dL (5.1 micromol/L), of the total bilirubin concentration [11,13].

Advances in laboratory methodology have demonstrated that the diazo method may not accurately reflect the concentration of conjugated and unconjugated bilirubin. Direct bilirubin overestimates the conjugated bilirubin concentration because a fraction of unconjugated bilirubin (about 10 to 15 percent) also gives a direct reaction using the van den Bergh method. There are several other potential sources of error. Endogenous substances, such as plasma lipids, and drugs, such as propranolol, interfere with the diazo reaction, and can produce unreliable results. Fortunately, these interactions are only significant when the bilirubin concentration is normal or slightly elevated [14,15]. Bilirubin complexed to albumin (delta bilirubin) also may give a direct reaction [16,17]. (See 'Delta bilirubin' below.)

Alkaline methanolysis — A more accurate and sensitive quantification of bilirubin requires chromatographic analysis, such as high-performance liquid chromatography (HPLC) and reflectance fluorometry. One such method involves alkaline methanolysis of bilirubin followed by chloroform extraction of the bilirubin methyl esters. Separation of these esters is performed using HPLC and spectrophotometry [14,15]. Using HPLC, conjugated bilirubin accounts for approximately 4 percent of the total serum bilirubin [18].

Other HPLC-based methods are also available that do not require alkaline methanolysis, although globulins and other high-molecular weight proteins must be precipitated from serum before chromatography [19,20]. Highly accurate methods for direct analysis of free bilirubin in serum combine HPLC and thermal lens spectometry or diode array detection [21].

Other methods — Several other methods that use dry reagent chemistry have been reported. One method, which is used in many clinical chemistry laboratories, is based upon photographic film technology [19]. It can be automated and appears able to measure conjugated and unconjugated bilirubin accurately. This technique also has the advantage of being able to detect delta bilirubin, the fraction conjugated to albumin. A method using reagent-free visible-near-infrared spectroscopy has been proposed for rapid screening of large populations [22].

These newer techniques have added considerably to our understanding of bilirubin metabolism.

Virtually 100 percent of the serum bilirubin in healthy people, including those with Gilbert syndrome, is unconjugated. Conjugated hyperbilirubinemia occurs only in hepatobiliary diseases.

The observation that jaundiced patients with hepatobiliary diseases have lower serum bilirubin concentrations measured by non-diazo- than diazo-based methods suggests that there are additional diazo-positive compounds distinct from bilirubin in their circulation [15].

Monoglucuronides of bilirubin predominate over the diglucuronides in jaundiced patients with hepatobiliary disease.

Delta bilirubin — The ability to detect delta bilirubin has important clinical implications. This albumin-linked bilirubin fraction is formed in the serum when hepatic excretion of bilirubin glucuronides is impaired; as a result, it represents a significant fraction of total serum bilirubin in patients with cholestasis and hepatobiliary disorders [17]. In one series, for example, delta bilirubin constituted 8 to 90 percent of total bilirubin in patients with hepatocellular and cholestatic jaundice, but was undetectable in normal volunteers, neonates with physiologic jaundice, or those with Gilbert syndrome or hemolysis. Because of its covalent binding to albumin, the clearance of delta bilirubin is approximately the same as albumin rather than the short half-life of conjugated bilirubin that is not albumin bound (12 to 24 days versus 4 hours) [23].

Appreciation of the prolonged half-life of delta bilirubin has explained two observations in jaundiced patients that were previously not understood:

Because conjugated bilirubin is excreted in the urine, patients with conjugated hyperbilirubinemia develop bilirubinuria. However, some patients with conjugated hyperbilirubinemia do not exhibit bilirubinuria during the recovery phase of their disease because delta bilirubin persists longer than bilirubin and delta bilirubin is not excreted in the urine.

Late in the recovery phase of hepatobiliary disorders, virtually all the conjugated bilirubin may be in the albumin-linked form. As a result, elevated serum bilirubin levels decline more slowly than expected in some patients who otherwise appear to be recovering satisfactorily.

CLINICAL SIGNIFICANCE OF SERUM BILIRUBIN — The bilirubin normally present in serum reflects a balance between production and clearance. Thus, elevated serum bilirubin concentrations can be due to three causes which can sometimes coexist:

Overproduction of bilirubin

Impaired uptake, conjugation, or excretion of bilirubin

Backward leakage from damaged hepatocytes or bile ducts

An approach to patients who present with hyperbilirubinemia is presented separately (see "Diagnostic approach to the adult with jaundice or asymptomatic hyperbilirubinemia"). This section will review the clinical significance of hyperbilirubinemia.

Indication of the severity of hepatic dysfunction — Total serum bilirubin is not a sensitive indicator of hepatic dysfunction. Concentrations of serum bilirubin may be normal despite moderate to severe hepatic parenchymal injury or a partially or transiently obstructed bile duct.

This lack of sensitivity can be explained in part by the reserve capacity of the human liver to remove bilirubin. Studies of healthy people who were given infusions of unconjugated bilirubin, and observations in patients who have hemolysis, have demonstrated that the normal liver can remove at least twice the normal daily bilirubin load without the development of hyperbilirubinemia [24]. The reserve capacity may be even higher based upon the maximal rate of excretion of bilirubin in bile. The maximal daily excretion of bilirubin is approximately 55.2 mg/kg, which is more than 10 times greater than the average daily production [25].

Correlation of bilirubin concentration with jaundice — In a steady state, the serum bilirubin concentration usually reflects the intensity of jaundice and the amount of bilirubin pigment in the body. However, several factors can influence the relationship between serum bilirubin and the total body bilirubin content. The serum bilirubin concentration may be lowered transiently by salicylates, sulfonamides, or free fatty acids, which displace bilirubin from its attachment to plasma albumin, thereby enhancing transfer of the pigment into tissues [26]. In contrast, an increase in the serum albumin concentration (eg, due to volume contraction) may induce a temporary shift of bilirubin from tissue sites into the circulation [27].

Significance of the serum bilirubin concentration — The serum total bilirubin concentration is seldom of value in specifying the cause of jaundice in individual patients because values among the various causes of jaundice overlap considerably [28]. As general rules:

Uncomplicated hemolysis seldom causes a serum bilirubin value in excess of 5 mg/dL (85.5 micromol/L) [29].

Parenchymal liver disease and incomplete extrahepatic biliary obstruction due to calculi are associated with lower serum bilirubin concentrations than malignant obstruction of the bile duct.

Although few controlled studies have evaluated the usefulness of the level and duration of the serum bilirubin for determining disease prognosis, several general relationships appear to apply. The higher the serum bilirubin concentration in viral hepatitis, the greater the histologic evidence of hepatocellular damage and the longer the course of the disease [30]. Similarly a serum bilirubin concentration higher than 5 mg/dL (85.5 micromol/L) is associated with a poor prognosis in alcohol-associated hepatitis [31,32]. A rising bilirubin concentration suggests a poor prognosis in patients with primary biliary cholangitis [33].

However, the correlation of serum bilirubin concentration with disease outcome does not always hold true. As an example, patients may die of acute liver failure with only modest elevations of the serum bilirubin. Furthermore, conditions associated with excess bilirubin production (such as hemolysis) or decreased clearance (such as renal insufficiency) can result in hyperbilirubinemia out of proportion to the degree of hepatic dysfunction.

Value of fractionating the bilirubin — Elevated levels of unconjugated bilirubin usually result from overproduction or impaired uptake or conjugation of bilirubin. In contrast, conjugated hyperbilirubinemia is more commonly due to decreased excretion or backward leakage (as from an obstructed biliary system) and is usually a more sensitive indicator of hepatic dysfunction. The new, more precise methods for measuring serum bilirubin (see 'Measurement of serum bilirubin' above) have demonstrated that virtually 100 percent of the serum bilirubin in healthy people and those with Gilbert syndrome is unconjugated. Measurable amounts of conjugated bilirubin in serum using the most specific laboratory techniques are found only in hepatobiliary disease. However, many clinical laboratories continue to use less precise techniques, and thus report a normal range for conjugated bilirubin.

The major value of fractionating total serum bilirubin is for the detection of states characterized by unconjugated hyperbilirubinemia (table 2) (see "Classification and causes of jaundice or asymptomatic hyperbilirubinemia"). These diagnoses should be considered when the serum indirect bilirubin concentration is greater than 1.2 mg/dL (20.5 micromol/L) and the direct fraction constitutes less than 15 percent of the total serum bilirubin. Using the diazo method (see 'van den Bergh method' above), an increase in the direct bilirubin concentration above 0.3 mg/dL (5.1 micromol/L) or, using the more accurate techniques, a direct bilirubin concentration above 0.1 mg/dL (1.7 micromol/L) should raise suspicion for liver injury [15].

Fractionation of the serum bilirubin concentration in jaundiced patients does not permit accurate distinction between parenchymal (hepatocellular) and cholestatic (obstructive) jaundice. The accurate HPLC methods for measuring serum bilirubin demonstrate that unconjugated and conjugated bilirubin are both increased in hepatobiliary disease without a consistent difference in pattern [15]. Levels of both bilirubin monoglucuronide and diglucuronide are elevated, with the monoglucuronides predominating.

Urine bilirubin — The presence of bilirubin in the urine reflects direct hyperbilirubinemia and therefore underlying hepatobiliary disease. In contrast to conjugated bilirubin, unconjugated bilirubin is tightly bound to albumin; as a result, it is not filtered by the glomerulus or present in the urine.

Conjugated bilirubin may be found in the urine when the total serum bilirubin concentration is normal because the renal reabsorptive capacity for conjugated bilirubin is low and the methods used can detect urinary bilirubin concentrations as low as 0.05 mg/dL (0.9 micromol/L) [34]. Thus, bilirubinuria may be an early sign of liver disease, while the clearance of bilirubin from the urine may be an early sign of recovery, since, as noted above, delta bilirubin is protein-bound (thereby increasing its half-life compared with nonbound conjugated bilirubin and preventing its filtration across the glomerulus) [23].

SUMMARY AND RECOMMENDATIONS

Bilirubin is the catabolic product of heme metabolism, which is formed by the breakdown of heme present in hemoglobin, myoglobin, cytochromes, catalase, peroxidase, and tryptophan pyrrolase (figure 1). Eighty percent of the daily bilirubin production (250 to 400 mg) is derived from hemoglobin; the remaining 20 percent is contributed by other heme proteins and a small pool of free heme that turns over rapidly. Some of the bilirubin produced is then conjugated in the liver (figure 2).

The bilirubin normally present in serum reflects a balance between production and clearance. Thus, elevated serum bilirubin concentrations can be due to three causes that sometimes coexist (see 'Clinical significance of serum bilirubin' above):

Overproduction of bilirubin

Impaired uptake, conjugation, or excretion of bilirubin

Backward leakage from damaged hepatocytes or bile ducts

Total serum bilirubin is not a sensitive indicator of hepatic dysfunction. (See 'Indication of the severity of hepatic dysfunction' above.)

The level of the total serum bilirubin is seldom of value in specifying the cause of jaundice in individual patients because values among the various causes of jaundice overlap considerably. (See 'Significance of the serum bilirubin concentration' above.)

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