Assessment of Liver Function and Diagnostic Studies

Serum Bilirubin

• 1.

  Jaundice

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    Often the first evidence of liver disease

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    Clinically apparent when serum bilirubin exceeds 3 mg/dL; patient may notice dark urine or pale stool before conjunctival icterus

• 2.

  Metabolism

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    Bilirubin is a breakdown product of hemoglobin and, to a lesser extent, heme-containing enzymes; 95% of bilirubin is derived from senescent red blood cells.

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    After red blood cell breakdown in the reticuloendothelial system, heme is degraded by the enzyme heme oxygenase in the endoplasmic reticulum.

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    Bilirubin is released into blood and tightly bound to albumin; free or unconjugated bilirubin is lipid soluble, is not filtered by the glomerulus, and does not appear in urine.

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    Unconjugated bilirubin is taken up by the liver by a carrier-mediated process, attaches to intracellular storage proteins (ligands), and is conjugated by the enzyme uridine diphosphate (UDP)–glucuronyl transferase to form a diglucuronide and, to a lesser extent, a monoglucuronide.

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    Conjugated bilirubin is water soluble and thus appears in urine.

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    When serum levels of bilirubin glucuronides are elevated, some binding to albumin occurs (delta bilirubin) , leading to absence of bilirubinuria despite conjugated hyperbilirubinemia; this phenomenon explains delayed resolution of jaundice during recovery from acute liver disease until albumin-bound bilirubin is catabolized.

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    Conjugated bilirubin is excreted by active transport across the canalicular membrane into bile.

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    Bilirubin in bile enters the small intestine; in the distal ileum and colon, bilirubin is hydrolyzed by beta-glucuronidases to form unconjugated bilirubin, which is then reduced by intestinal bacteria to colorless urobilinogens; a small amount of urobilinogen is reabsorbed by the enterohepatic circulation and mostly excreted in the bile, with a smaller proportion undergoing urinary excretion.

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    Urobilinogens or their colored derivatives urobilins are excreted in feces.

• 3.

  Measurement of serum bilirubin

  • a.

    van den Bergh reaction

    • ▪

      Total serum b ilirubin represents all bilirubin that reacts with diazotized sulfanilic acid to form chromogenic pyrroles within 30 minutes in the presence of alcohol (an accelerating agent).

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      Direct serum bilirubin is the fraction that reacts with the diazo reagent in an aqueous medium within 1 minute and corresponds to conjugated bilirubin.

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      Indirect serum bilirubin represents unconjugated bilirubin and is determined by subtracting the direct reacting fraction from the total bilirubin level.

  • b.

    More specific methods (e.g., high-pressure liquid chromatography) demonstrate that the van den Bergh reaction often overestimates the amount of conjugated bilirubin; however, the van den Bergh method remains the standard test.

• 4.

  Classification of hyperbilirubinemia

  • a.

    Unconjugated (bilirubin nearly always <7 mg/dL)

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      Overproduction (presentation to liver of bilirubin load that exceeds hepatic capacity for uptake and conjugation): Hemolysis, ineffective erythropoiesis, resorption of hematoma

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      Defective uptake and storage of bilirubin: Gilbert syndrome (idiopathic unconjugated hyperbilirubinemia)

  • b.

    Conjugated

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      Hereditary: Dubin-Johnson and Rotor syndromes, bile transport protein defects

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      Cholestasis (Bilirubin is not a sensitive test of hepatic dysfunction.)

      • –

        Intrahepatic: Cirrhosis, hepatitis, primary biliary cholangitis, drug induced

      • –

        Extrahepatic biliary obstruction: Choledocholithiasis, stricture, neoplasm, biliary atresia, sclerosing cholangitis

  • c.

    Very high bilirubin levels

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      >30 mg/dL: Usually signifies hemolysis plus parenchymal liver disease or biliary obstruction; urinary excretion of conjugated bilirubin may help prevent even higher levels of hyperbilirubinemia; renal failure contributes to hyperbilirubinemia.

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      >60 mg/dL: Seen in patients with hemoglobinopathies (e.g., sickle cell disease) in whom obstructive jaundice or acute hepatitis develops.

  • d.

    The diagnostic approach to the evaluation of an isolated serum bilirubin level is shown in Fig. 1.1 .

    

• 5.

  Urine bilirubin and urobilinogen

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    Bilirubinuria indicates an increase in serum conjugated (direct) bilirubin.

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    Urinary urobilinogen (rarely measured now) is found in patients with hemolysis (increased production of bilirubin), gastrointestinal hemorrhage, or hepatocellular disease (impaired removal of urobilinogen from blood).

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    Absence of urobilinogen from urine suggests interruption of the enterohepatic circulation of bile pigments, as in complete bile duct obstruction.

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    Urobilinogen detection and quantification add little diagnostic information to the evaluation of hepatic dysfunction.

Serum Aminotransferases ( Table 1.1 )

• 1.

  These intracellular enzymes are released from injured hepatocytes and are the most useful marker of hepatic injury (inflammation or cell necrosis).

  • a.

    Aspartate aminotransferase (AST, serum glutamic oxaloacetic transaminase [SGOT])

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      Found in cytosol and mitochondria

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      Found in liver as well as skeletal muscle, heart, kidney, brain, and pancreas

  • b.

    Alanine aminotransferase (ALT, serum glutamic pyruvic transaminase [SGPT])

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      Found in cytosol

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      Highest concentration in liver (more sensitive and specific than AST for liver inflammation and hepatocyte necrosis)

  TABLE 1.1

  Causes of Elevated Serum Aminotransferase Levels ^a

  Mild Elevation (<5× normal)	Marked Elevation (>15× normal)
  Hepatic: ALT predominant Chronic viral hepatitis Acute viral hepatitis (A–E, EBV, CMV) NAFLD Hemochromatosis DILI Autoimmune hepatitis Alpha-1 antitrypsin deficiency Wilson disease Celiac disease Glycogenic hepatopathy Hepatic: AST predominant Alcohol-related liver injury (AST/ALT >2:1) Cirrhosis Nonhepatic Strenuous exercise Hemolysis Myopathy Thyroid disease Macro-AST	Acute viral hepatitis (A–E, herpes) DILI Ischemic hepatitis Autoimmune hepatitis Wilson disease Acute bile duct obstruction Acute Budd-Chiari syndrome Hepatic artery ligation

  ALT , Alanine aminotransferase; AST , aspartate aminotransferase; CMV , cytomegalovirus; DILI , drug-induced liver injury; EBV , Epstein-Barr virus; NAFLD , nonalcoholic fatty liver disease.

  a Almost any liver disease may be associated with ALT levels 5 times to 15 times normal.

• 2.

  Clinical usefulness

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    Normal levels of ALT are up to ~30 U/L in men and up to ~19 U/L in women.

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    Levels increase with body mass index (and particularly with trunk fat) and correlate with serum triglyceride, glucose, insulin, and leptin levels and possibly inversely with serum vitamin D levels. There is controversy as to whether levels correlate with the risk of coronary artery disease and mortality.

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    Levels may rise acutely with a high caloric meal or ingestion of acetaminophen 4 g/day; coffee appears to lower levels.

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    Aminotransferase elevations are often the first biochemical abnormalities detected in patients with viral, autoimmune, or drug-induced hepatitis; the degree of elevation may correlate with the extent of hepatic injury but is generally not of prognostic significance.

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    In alcoholic hepatitis, the serum AST is usually no more than 2 to 10 times the upper limit of normal, and the ALT is normal or nearly normal, with an AST:ALT ratio >2; relatively low ALT levels may result from a deficiency of pyridoxal 5-phosphate, a necessary cofactor for hepatic synthesis of ALT. In contrast, in nonalcoholic fatty liver disease, ALT is typically higher than AST until cirrhosis develops.

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    Aminotransferase levels may be higher than 3000 U/L in acute or chronic viral hepatitis or drug-induced liver injury; in acute liver failure or ischemic hepatitis (shock liver), even higher values (>5000 U/L) may be found.

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    Mild-to-moderate elevations of aminotransferase levels are typical of chronic viral hepatitis, autoimmune hepatitis, hemochromatosis, alpha-1 antitrypsin deficiency, Wilson disease, and celiac disease.

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    In obstructive jaundice, aminotransferase values are usually lower than 500 U/L; rarely, values may reach 1000 U/L in acute choledocholithiasis or 3000 U/L in acute cholecystitis, followed by a rapid decline to normal.

• 3.

  The approach to the patient with a persistently elevated ALT level is shown in Fig. 1.2 .

  

• 4.

  Abnormally low aminotransferase levels have been associated with uremia and chronic hemodialysis; chronic viral hepatitis in this population may not result in aminotransferase elevation.

Serum Alkaline Phosphatase

• 1.

  Hepatic ALP is one of several ALP isoenzymes found in humans and is bound to the hepatic canalicular membrane; various laboratory methods are available for its measurement, and comparison of results obtained by different techniques may be misleading.

• 2.

  This test is sensitive for detection of biliary tract obstruction (a normal value is highly unusual in significant biliary obstruction); interference with bile flow may be intrahepatic or extrahepatic.

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    An increase in serum ALP results from increased hepatic synthesis of the enzyme, rather than leakage from bile duct cells or failure to clear circulating ALP; because it is synthesized in response to biliary obstruction, the ALP level may be normal early in the course of acute cholangitis when the serum aminotransferases are already elevated.

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    Increased bile acid concentrations may promote the synthesis of ALP.

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    Serum ALP has a half-life of 17 days; levels may remain elevated up to 1 week after relief of biliary obstruction and return of the serum bilirubin level to normal.

• 3.

  Isolated elevation of alkaline phosphatase

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    This may indicate infiltrative liver disease: Tumor, abscess, granulomas, or amyloidosis.

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    High levels are associated with biliary obstruction, sclerosing cholangitis, primary biliary cholangitis, immunoglobulin (Ig) G4–associated cholangitis, acquired immunodeficiency syndrome, cholestatic drug reactions, and other causes of vanishing bile duct syndrome; in critically ill patients with sepsis, high levels may result from secondary sclerosing cholangitis from ischemia with rapid progression to cirrhosis.

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    Nonhepatic sources of ALP are bone, intestine, kidney, and placenta (different isoenzymes); elevations are seen in Paget disease of the bone, osteoblastic bone metastases, small bowel obstruction, and normal pregnancy.

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    A hepatic origin of an elevated ALP level is suggested by simultaneous elevation of either serum gamma-glutamyltranspeptidase (GGTP) or 5 ʹ -nucleotidase (5NT).

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    Hepatic ALP is more heat stable than bone ALP. The degree of overlap makes this test less useful than GGTP or 5NT.

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    The diagnostic approach to an isolated elevated ALP level is shown in Fig. 1.3 .

    

• 4.

  Mild elevations of serum ALP are often seen in hepatitis and cirrhosis.

• 5.

  Low serum levels of ALP may occur in hypothyroidism, pernicious anemia, zinc deficiency, congenital hypophosphatasia, and fulminant Wilson disease.

Gamma-Glutamyltranspeptidase

• 1.

  Although present in many different organs, GGTP is found in particularly high concentrations in the epithelial cells lining biliary ductules.

• 2.

  It is a very sensitive indicator of hepatobiliary disease but is not specific. Levels are elevated in other conditions, including renal failure, myocardial infarction, pancreatic disease, and diabetes mellitus.

• 3.

  GGTP is inducible, and thus levels may be elevated by ingestion of phenytoin or alcohol in the absence of other clinical evidence of liver disease.

• 4.

  Because of its long half-life of 26 days, GGTP is limited as a marker of surreptitious alcohol consumption.

• 5.

  Its major clinical use is to exclude a bone source of an elevated serum ALP level.

• 6.

  Many patients with isolated serum GGTP elevation have no other evidence of liver disease; an extensive evaluation is usually not warranted. Patients should be retested after avoiding alcohol and other hepatotoxins for several weeks.

5’-Nucleotidase

• 1.

  5NT is found in the liver in association with canalicular and sinusoidal plasma membranes.

• 2.

  Although 5NT is distributed in other organs, serum levels are believed to reflect hepatobiliary release by the detergent action of bile salts on plasma membranes.

• 3.

  Serum 5NT levels correlate well with serum ALP levels; an elevated serum 5NT level in association with an elevated ALP level is specific for hepatobiliary dysfunction and is superior to GGTP in this regard.

Lactate Dehydrogenase

Measurement of lactate dehydrogenase (LDH) and the more specific isoenzyme LDH5 adds little to the evaluation of suspected hepatic dysfunction. High levels of LDH are seen in hepatocellular necrosis, ischemic hepatitis, cancer, and hemolysis. The ALT/LDH ratio may help differentiate acute viral hepatitis (≥1.5) from ischemic hepatitis and acetaminophen toxicity (<1.5).

Serum Proteins

Most proteins circulating in plasma are produced by the liver and reflect its synthetic capacity.

• 1.

  Albumin

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    Albumin accounts for 75% of serum proteins.

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    Its half-life is approximately 3 weeks.

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    The concentration in blood depends on the albumin synthetic rate (normal, 12 g/day) and plasma volume.

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    Hypoalbuminemia may result from expanded plasma volume or decreased albumin synthesis. It is frequently associated with ascites and expansion of the extravascular albumin pool at the expense of the intravascular albumin pool. Hypoalbuminemia is common in chronic liver disease (an indicator of severity); it is less common in acute liver disease. It is not specific for liver disease and may also reflect glomerular or gastrointestinal losses.

• 2.

  Globulins

  • a.

    Globulins are often increased nonspecifically in chronic liver disease.

  • b.

    The pattern of elevation may suggest the cause of the underlying liver disease.

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      Elevated IgG: Autoimmune hepatitis

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      Elevated IgM: Primary biliary cholangitis

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      Elevated IgA: Alcoholic liver disease

• 3.

  Coagulation factors

  • a.

    Most coagulation factors are synthesized by the liver, including factors I (fibrinogen), II (prothrombin), V, VII, IX, and X and have much shorter half-lives than that of albumin.

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      Factor VII decreases first in liver disease because of its shortest half-life, followed by factors X and IX.

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      Factor V is not vitamin K dependent, and its measurement can help distinguish vitamin K deficiency from hepatocellular dysfunction in a patient with prolonged prothrombin time. Serial measurement of factor V levels has been used to assess prognosis in acute liver failure; a value <20% of normal portends a poor outcome without liver transplantation.

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      Measurement of factor II (des-gamma-carboxyprothrombin) has also been used to assess liver function. Elevated levels are found in cirrhosis and hepatocellular carcinoma (HCC) and in patients taking warfarin, a vitamin K antagonist. Administration of vitamin K results in normalization of des-gamma-carboxyprothrombin in patients taking warfarin but not in those with cirrhosis.

  • b.

    The prothrombin time is useful in assessing the severity and prognosis of acute liver disease. The one-stage prothrombin time described by Quick measures the rate of conversion of prothrombin to thrombin after activation of the extrinsic coagulation pathway in the presence of a tissue extract (thromboplastin) and calcium (Ca ⁺⁺ ) ions. Deficiency of one or more of the liver-produced factors results in a prolonged prothrombin time.

  • c.

    Prolongation of the prothrombin time in cholestatic liver disease may result from vitamin K deficiency.

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      Explanations for a prolonged prothrombin time apart from hepatocellular disease or vitamin K deficiency include consumptive coagulopathies, inherited deficiencies of a coagulation factor, or medications that antagonize the prothrombin complex.

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      Vitamin K deficiency as the cause of a prolonged prothrombin time can be excluded by administration of vitamin K 10 mg; intravenous administration can cause severe reactions, and the oral route is preferable, if possible. (Subcutaneous administration is not recommended because of erratic absorption.) Correction or improvement of the prothrombin time by at least 30% within 24 hours implies that hepatic synthetic function is intact.

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      The international normalized ratio (INR) is used to standardize prothrombin time determinations performed in different laboratories; however, the results are less consistent in patients with liver disease than in those taking warfarin unless liver-disease controls are used.

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      The prothrombin time and INR correlate with the severity of liver disease but not with the risk of bleeding because of counterbalancing decreases in levels of anticoagulant factors (e.g., proteins C and S, antithrombin) and enhanced fibrinolysis in patients with liver disease.

Antipyrine Clearance

• 1.

  Antipyrine is metabolized by cytochrome P-450 oxygenase with good absorption after oral administration and elimination entirely by the liver.

• 2.

  In chronic liver disease, good correlation exists between prolongation of the antipyrine half-life and disease severity as assessed by the Child-Turcotte-Pugh score (see Chapter 11 ).

• 3.

  Clearance of antipyrine is less impaired in acute liver disease and obstructive jaundice than in chronic liver disease.

• 4.

  Disadvantages of this test include its long half-life in serum, which requires multiple blood sampling, poor correlation with in vitro assessment of hepatic microsomal capacity, and alteration of antipyrine metabolism by increased age, diet, alcohol, smoking, and environmental exposure.

Aminopyrine Breath Test

• 1.

  This test is based on detection of [ ¹⁴ C]O ₂ in breath 2 hours after an oral dose of [ ¹⁴ C]dimethyl aminoantipyrine (aminopyrine), which undergoes hepatic metabolism.

• 2.

  Excretion is diminished in patients with cirrhosis as well as those with acute liver disease.

• 3.

  The test has been used to assess prognosis in patients with alcoholic hepatitis and in cirrhotic patients who are undergoing surgery.

• 4.

  A limitation of the aminopyrine breath test is its lack of sensitivity in hepatic dysfunction resulting from cholestasis or extrahepatic obstruction.