Laboratory evaluation of the hepatobiliary system centers on measurements of: 1) hepatocyte plasma membrane integrity, 2) measurements of the detoxifying and excretory functions of the hepatobiliary system, and 3) measurements of the synthetic capacity of hepatocytes.

With hepatocyte or biliary tract disease, many cellular enzymes are released that enter the circulation. Enzymes indicative of hepatocyte disease are alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Alkaline phosphatase (ALP) elevations relate to biliary tract disease (Table 16–1).

Enzyme concentrations are usually measured by determining the enzyme activity in serum or plasma. Such measurements are reported as units per liter or international units per liter, where the unit is an activity measurement (eg, the rate of appearance of product or disappearance of substrate per unit time).

Normally, the healthy plasma membrane and various organelles contain (eg, “hold”) enzymes within the cell. An elevated enzyme level in the blood suggests organ dysfunction because of a functional or anatomic disruption in the plasma membrane. One way to assess the degree of elevation of an enzyme is to calculate the ratio of the patient’s enzyme concentration relative to the upper limit of the reference interval. For example, if the upper limit of the reference interval for ALT were 40 U/L and the patient’s ALT was 120 U/L, the patient’s ALT would be said to be “3 times above the upper limit of normal.”

While not specific for hepatocytes, elevations of ALT and AST are characteristic of hepatocellular disease. The major sources of ALT include the liver and the kidney. Lesser amounts are released from skeletal and cardiac muscle. AST is also found in these organs. ALT is exclusively localized in the cell cytoplasm. AST is located in the cytoplasm and mitochondria. However, AST derived from the cytoplasm and mitochondria cannot be distinguished through clinical laboratory testing. ALT is more specific for the liver than AST. Usually ALT and AST rise in tandem in liver disease states. Although there is more AST in hepatocytes than ALT, ALT is metabolized more slowly than AST accounting for similar concentrations of these enzymes in the patient’s plasma as released from the liver.

One condition where AST is often elevated to a greater extent than ALT is in chronic liver disease resulting from chronic alcohol abuse. People with alcoholism are not uncommonly pyridoxine deficient because of deficient dietary intake of this vitamin. While both AST and ALT are pyridoxine dependent for their biochemical activity, ALT is more dependent on pyridoxine than AST. Thus, a rise in the measured ALT may not be as great as the rise in measured AST because ALT activity suffers more from pyridoxine deficiency than does AST. If the AST to ALT ratio is greater than 2 in the setting of chronic liver disease, alcoholic liver disease is strongly suggested. With cirrhosis of any etiology, enzyme elevations may be modest, or their concentrations may be surprisingly normal, reflecting a marked loss in hepatocyte mass and, thereby, a loss of enzymes within the liver.

In the past, lactate dehydrogenase (LD) was also regularly employed as a marker of hepatocellular disease. (Note: The older abbreviation for lactate dehydrogenase was “LDH.”) However, LD is not favored for routine evaluation of the hepatocyte integrity currently because LD is released with injury of many different tissues. Both ALT and AST are more specific for liver disease or injury than LD.

Measurement of LD isoenzymes is possible, but there are more informative tests that can be ordered to evaluate specific organ dysfunction. LD is composed of 4 subunits. The subunits are H (for heart) and M (for muscle). If required, LD isoenzymes can be determined by electrophoresis. The subunit composition and major sources of each of the 5 isoenzymes are listed in Table 16–2. The LD4 isoenzyme provides no specific clinically useful information.

If the total LD is increased in a patient with suspected liver disease, and the patient lacks skeletal muscle and prostate disease, it is expected that LD5 will be elevated because of the liver disease. The enzyme marker of choice for the evaluation of skeletal muscle injury or disease is creatine kinase (CK). If the CK is normal in the setting of an elevated LD5, skeletal muscle is not likely to be the source of the elevated LD5.

Biliary tract disease produces relatively greater increases in ALP than increases in ALT, AST, or LD. ALP is associated with the plasma membrane of hepatocytes adjacent to the biliary canaliculus. Obstruction or inflammation of the biliary tract results in an increased concentration of the ALP in the circulation. Similar to ALT and AST, ALP is not specific for biliary tract disease. ALP is released by osteoblasts, the ileum, and the placenta. ALP is elevated: 1) in children 2- to 3-fold over adults because the child’s skeleton is growing, 2) with bone disease involving osteoblasts (eg, metastatic cancer or following a fracture), 3) in hyperparathyroidism where parathyroid hormone stimulates osteoblasts through a series of steps that enhances bone resorption (eg, parathyroid adenoma, hyperplasia, or secondary hyperparathyroidism from vitamin D deficiency or renal disease), 4) in cases of ileal disease, and 5) during the third trimester of pregnancy because the placental isoenzyme is elevated.

When the etiology of the elevated ALP is unclear, in the past ALP isoenzymes were determined. However, there are many technical problems with these assays. Today it has proven more pragmatic to measure other biliary tract enzyme markers such as gamma-glutamyl transpeptidase (GGT; aka gamma-glutamyltransferase) or 5’-nucleotidase (5’-NT). The proximal convoluted tubule of the kidney, the liver, the pancreas, and the intestine are sources of GGT, in decreasing order of tissue concentration. Within the cell. GGT is located in microsomes and along the biliary tract plasma membrane, GGT is more commonly measured than 5’-NT because GGT testing is widely available on a variety of laboratory instruments. GGT is typically not elevated with bone disease. Combined elevations of ALP and GGT are compatible with biliary tract disease. However, if the ALP is elevated to a far greater extent than the GGT (or the GGT is normal), ALP sources other than the biliary tract, such as bone, must be investigated. GGT elevations occur in response to alcohol use and anticonvulsants, as GGT is induced by such agents. While there is no specific biochemical test to prove that a patient suffers from alcohol abuse, carbohydrate-deficient transferrin levels can be elevated in patients suffering from alcoholism.

Using the information presented, one can interpret of liver enzyme elevations in patients with suspected liver disease. If the relative increase in ALT or AST over the upper limit of normal exceeds the relative increase in ALP over the upper limit of normal, the liver disease is predominantly hepatocellular as opposed to biliary tract.

Causes of acute hepatocellular disease include (Table 16–3) viral hepatitis (eg, hepatitis A, B, or C), alcoholic hepatitis, toxic injury (eg, acetaminophen poisoning), and ischemic injury (eg, hypotension). In cases of ischemic injury or toxic injury following an acute toxic ingestion, the ALT and AST levels can rise and peak within 24 hours of the precipitating event. Less common causes of acute liver disease include hepatitis due to hepatitis D, hepatitis E, cytomegalovirus (CMV), Epstein–Barr virus (EBV), and herpes virus; autoimmune hepatitis (marked by positivity for antinuclear antibodies [ANA], smooth muscle autoantibodies [ASMA], and/or liver–kidney microsome autoantibodies [anti-LKM₁ autoantibodies] and negative antimitochondrial autoantibodies [AMA]); Wilson disease; and liver disease of pregnancy. Three forms of liver disease in pregnancy include fatty liver, intrahepatic cholestasis, and hepatic dysfunction associated with toxemia (eg, part of the HELLP syndrome: hemolysis, elevated LFTs [eg, enzymes], and low platelets).

Chronic hepatocellular disease is diagnosed when liver disease is present for more than 6 months (Table 16–3). Causes of chronic hepatocellular disease include hepatitis B or C, drug toxicity (eg, statins, sulfonamides, or INH), alcoholic liver disease, nonalcoholic fatty liver (NAFL), inborn errors of metabolism, and autoimmune hepatitis. NAFL is one of the most common causes of nonviral and nonalcoholic liver disease. It can progress to nonalcoholic steatohepatitis (NASH), cirrhosis, liver failure, and even hepatocellular carcinoma in some cases. Inborn errors causing chronic liver disease encompass hemochromatosis, alpha-1 antitrypsin deficiency, Wilson disease, glycogen storage disease (GSD), and Gaucher disease.

The AST to ALT ratio can be used to suggest alcoholic liver disease. One can argue that excluding the setting of alcoholism, hepatocellular disease can be adequately assessed with the measurement of ALT alone. However, it is common medical practice to measure both enzymes, and the enzyme measurements are rapidly available and can be performed at low cost in modern automated laboratories.

If the relative increase in ALP over the upper limit of normal exceeds the relative increase in ALT or AST over the upper limit of normal, the liver disease predominantly involves the biliary tract (Table 16–4). A major manifestation of obstructive biliary tract disease is an elevated bilirubin concentration. Clinical jaundice results when the total bilirubin exceeds 2 to 3 mg/dL.

A major biochemical responsibility of the liver is to metabolize toxins, drugs, and biologic end products and excrete many of the resulting metabolites into the bile. The easiest endogenous end product to assess is the bilirubin concentration in the plasma. Bilirubin is predominantly derived from hemoglobin in the normal turnover of red blood cells, and to a lesser extent, from myoglobin in muscle. Red blood cells normally circulate for approximately 120 days. Red blood cell senescence and destruction in monocytes/macrophages, primarily in the spleen, releases hemoglobin from red blood cells. Within the phagocyte, hemoglobin is then metabolized to biliverdin and finally to bilirubin. The bilirubin then enters the circulation. This form of bilirubin (ie, “unconjugated” bilirubin) is relatively insoluble in water and is transported to the hepatocyte bound to albumin. It is not excreted in the urine. Unconjugated bilirubin is normally taken up into hepatocytes via a transport system. Inside the hepatocyte via the action of UDP-glucuronyl transferase, either 1 or 2 glucuronide molecules are conjugated to bilirubin, making the bilirubin water soluble. Conjugated bilirubin is bilirubin monoglucuronide or bilirubin diglucuronide. Conjugated bilirubin is then transported across the plasma membrane into the bile canaliculi along with bile via multiple drug resistance (MDR) transporter proteins. If the concentration of either conjugated or unconjugated bilirubin rises pathologically, the skin and sclera can develop a yellowish color, termed jaundice. With marked elevations in bilirubin, patients may acquire a green hue. Pathologic elevations in water-soluble bilirubin (eg, conjugated bilirubin) can lead to bilirubin excretion in the urine (bilirubinuria), causing the urine to develop a yellow-brown or green-brown color.

Bilirubin is most often measured by reacting the patient’s serum or plasma with Ehrlich reagent that includes a diazo compound. The conjugated fraction reacts most rapidly with the reagent because the conjugated fraction is water soluble. This is termed “direct acting,” or more commonly, “direct” bilirubin. To measure total bilirubin, solubilizing agents must be added to the serum or plasma to enhance the reaction of the water-insoluble bilirubin (ie, unconjugated bilirubin) with the reagents. Caffeine or benzoate can be used for this purpose. Because only direct and total bilirubin can be measured, indirect (unconjugated bilirubin) is calculated as the difference between the total and the direct bilirubin. While the terms “direct” and “conjugated” are used synonymously just as the terms “indirect” and “unconjugated” bilirubin are used synonymously, it should be noted that these are approximations. In fact, direct bilirubin measures 70% to 90% of the conjugated bilirubin, delta bilirubin (biliprotein, see below), and 5% to 10% of the unconjugated bilirubin.