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Author: Marshall Kaplan, M.D.
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1. Objectives: Liver Function Tests

  1. To be able to describe what blood tests are checked to evaluate patients with liver disease or with suspected liver disease.

  2. Understand the serum protein tests, enzyme tests, and immunological tests that are used in the evaluation of liver disease.

  3. Understand conjugated and unconjugated bilirubin.

  4. Understand the importance of the prothrombin time and albumin in the assessment of hepatic synthetic function.

2. Purpose

  1. Evaluation of Jaundice and Hepatic Enlargement

    1. Types of intrinsic liver disease

      1. Hepatocellular: viral or drug hepatitis

      2. Infiltrative: tumor, granuloma, “metabolic” (fat, glycogen, iron, copper), amyloid

      3. Cirrhosis: mixed hepatocellular disease and “infiltration” by scar tissue (many types); consequence of chronic liver disease

      4. Intrahepatic cholestasis: drug effect, primary biliary cirrhosis

    2. Types of extrahepatic bile duct obstruction – with or without cholangitis (infection)

      1. Gallstones

      2. Tumor

      3. Stricture, sclerosing cholangitis

      4. Inflammation (pancreatitis)

    3. Vascular cause of hepatic dysfunction

      1. Shock

      2. Left-and right-sided heart failure

      3. Budd-Chiari syndrome

    4. Abnormalities of bilirubin metabolism

      1. Hemolysis – compensated or uncompensated

      2. Defects in bilirubin conjugationa.

        1. Inherited lack of glucuronyl transferase (Crigler-Najjar syndrome)

        2. Delay in maturation of glucuronyl transferase in neonates

        3. Neonatal jaundice in breast-fed infants; cause uncertain but may be due to enhanced B-glucuronidase activity in the intestine of nursing infants causing de-conjugation of bilirubin-glucuronide

        4. Impaired uptake or conjugation of bilirubin by hepatocyte (Gilbert’s syndrome). Occurs in 3-6% of healthy adults, more men than women

      3. Defect in excretion of conjugated bilirubin – Dubin-Johnson and Rotor syndromes

      4. “Shunt” bilirubinemia – I. Bilirubin formation bypasses normal RBC hemoglobin (pernicious anemia, thalassemia)

  2. Detection of Systemic Disease by Liver Involvement

    1. Tumor – metastatic or primary → may cause isolated elevation of Lymphoma, leukemia; → serum alkaline phosphates

    2. Granulomatous disease – sarcoid, Tbc, fungi, brucella.

    3. “Collagen disease;” SLE, polymyaliga rheumatica; polyarteritis; amyloid (rheumatoid arthritis, multiple myeloma)

    4. Q fever, herpes simplex (children), parasites, Coxsackie, cytomegalovirus, toxoplasmosis, syphilis (rare)

    5. Graft versus host disease in bone marrow transplantation

    6. Acute and chronic heart failure

3. Limitations of Liver Function Tests

  1. Lack specificity

  2. Lack sensitivity

  3. Must be done in conjunction with other tests

  4. Helpful but rarely diagnostic

4. Liver Function Tests

  1. Serum proteins

    1. Albumin: synthesized by parenchymal cells; about 15-20 g/d. Maximum is 40 g/d. Normal half-life (t ½) is 14-20 days or approximately 4% degraded per day. Synthesis stimulated by low serum oncotic pressure; synthesis inhibited by malnutrition, interleukin-1, TNF. Serum levels may be decreased in chronic liver diseases such as cirrhosis, but also in non-hepatic chronic inflammatory diseases.

    2. Globulins: Many types. Immunoglobulins synthesized by plasma cells; alpha and beta by hepatocyte and intestinal mucosal cells. Electrophoresis separates globulins into components; gamma increased diffusely in hepatocellular diseases, cirrhosis, and particularly in autoimmune chronic hepatitis.

    3. Specific serum proteins:

      1. Ceruloplasmin: a blue copper binding protein in serum that functions as an oxidase. Normal values are between 25-35 mg/100 ml. Patients with Wilson’s disease have low values, often between 0-10 mg/100 ml. Patients with Wilson’s disease and serious liver involvement often will have higher-than-expected values, between 10 and 20 mg/100ml. Between 10 and 20 mg/100ml is a gray area and will include patients with disorders other than Wilson’s disease as well as some heterozygotes for Wilson’s.

      2. Alpha-1-globulin: this protein is measured by various electrophoretic techniques. Approximately 90% of alpha-1-globulin is alpha-1-antitrypsin. Therefore, marked diminution or absence of alpha-1-globulin in serum should make one think of homozygous alpha-1-antitrypsin deficiency, a relatively rare type of liver and lung disease.

      3. Ferritin: an iron binding protein that is measurable by radioimmunoassay. Normal ranges are approximately 20-200 mcg/L in males and 10-150 mcg/L in females. Values are elevated in iron storage disease such as hemochromatosis. Ferritin elevation in serum is nonspecific. Any type of liver cell injury, e.g., hepatitis, may elevate the serum ferritin to levels equal to or higher than those seen in hemochromatosis.

  2. Serum Enzymes

Aminotransferases (formerly called transaminases): dependent on vitamin B6 (pyridoxal phosphate) cofactor.
AST (aspartate aminotransferase) or SGOT (serum glutamate oxylacetate transaminase): L-glutamic acid + oxaloacetic acid → alpha-ketoglutarate + L-aspartic acid. Normal value, <35 International Units (IU). Primarily found in liver but also in heart and muscle, most in mitochondria, some in cytoplasm.
ALT (alanine aminotransferase) or SGPT (serum glutamate pyruvate transaminase): L-glutamic acid + pyruvic acid → alpha-ketoglutarate + L-alanine. Normal, < 25 IU. Elevation due to release from damaged or necrotic hepatocytes; extremely high values are found in hepatitis, acute liver congestion, toxic liver disease (in the 1,000-3,000 range). Elevation usually less than 400 units in bile duct obstruction and infiltrative diseases unless acute cholangitis present.
Serum AST and ALT levels are almost always less than 300 units in alcoholic liver disease and AST levels are higher than ALT levels. The opposite (ALT > AST) occurs in most other liver diseases.

  1. LDH (lactate dehydrogenase): pyruvic acid + NADH + H+ → lactic acid + NAD+; exists in five electrophoretically distinct forms, with each organ having characteristic proportion of isoenzymes – can separate liver LDH from cardiac LDH. Liver LDH has less electrophoretic mobility than cardiac LDH and is not inhibited by sulfite. Elevation due to release from necrotic cells. Much less useful than AST or ALT in diagnosis of liver disease.

  2. Alkaline phosphatase: many different units were once used. Now most hospitals use International Units.

    1. International Units (normal, 20-125 units). Paranitrophenylphosphate → paranitrophenol + Pi (inorganic phosphate); assay P-nitrophenol at alkaline pH. Note: normal values vary from hospital to hospital.

    2. Source – liver, bone, kidney, intestine, and placenta in third trimester of pregnant women. Can separate liver, bone and intestinal alkaline phosphatase electrophoretically. Can estimate intestinal alkaline phosphatase by selective inhibition with L-phenylaline and bone phospatase by its sensitivity to heating at 56 C or by incubation in 3M urea. Genetically, there are two major alkaline phosphatases that differ in amino acid sequence: unspecific alkaline phosphatase and intestinal alkaline phosphatase. Unspecific alkaline phosphatases are due to different types of carbohydrates linked to the peptide backbone.

    3. Basis for serum elevation:

      1. Liver disease – overproduction of alkaline phosphatase by liver and regurgitation into blood. Highest values in bile duct obstruction and some drug toxicities.

      2. Bone disease – overproduction by bone.

    4. The liver does not excrete serum alkaline phosphatase from serum into bile, but there is a small amount of alkaline phosphatase activity in bile.

  3. 5’-nucleotidase: adenosine-5’-monophosphate → adenosine + Pi – measure Pi (normal, 0.2-3.2 Bodansky Units); rarely elevated in bone disorders – frequently parallels alkaline phosphatase elevation in biliary tract disorders. Used to confirm liver origin of an elevated serum alkaline phosphatase.

  4. Gamma-glutamyl transpeptidase: A membrane-bound enzyme in bile duct cells and perhaps in hepatocytes that is elevated in almost any type of liver disease. However, it may be selectively and disproportionately elevated in alcoholic liver disease. It is also greatly elevated in bile duct obstruction and tends to behave like the serum alkaline phosphatase and 5’nucleotidase in this disorder. Like the 5’-nucleotidase, it is not elevated in bone diseases.

5. Carbohydrate Metabolism

  1. Glucose – glycogen normally 4% liver weight (60-70 g). A 70-kg man uses roughly 12 g gluc/hr. Serum glucose rarely depressed in liver disease unless it is far advanced and imminently fatal.

  2. Galactose tolerance – a relatively quantitative test of hepatocellular function but rarely used; dependent on conversion of gal → glu; galactose-1-p + UDP glucose → UDP gal + gal-1-P; UDP gal → UDP glucose.

6. Lipid Metabolism

  1. Phospholipids – increased in bile duct obstruction, particularly in primary biliary cirrhosis.

  2. Triglycerides, free fatty acids – usually unchanged but for marked, transient increase in acute pancreatitis and increase in obstructive jaundice.

  3. Cholesterol – in serum and RBC membranes, about 60% is esterified with fatty acids; in other human tissue, only 20-30% esterified, except for nervous tissue (myelin) and adrenal where almost all is free. Serum cholesterol esters diminished in hepatocellular disease and elevated in obstruction; HDL cholesterol is also elevated in obstructive liver disease.

  4. Bile acids – the total body pool is 3-5g; large enterohepatic circulation; serum levels rise in obstruction; bound in gut with non-absorbable exchange resin cholestyramine. Tissue levels can be depleted with oral cholestyramine, 8-16 g/day. Bile salts are deconjugated in GI tract by the gut flora and deconjugated bile acids may cause steatorrhea in bacterial overgrowth syndromes.

7. Detoxification

  1. NH3 – fixed in liver primarily as urea: metabolic pathways for NH3 utilization include:

    1. NH3 and CO2 + ATP → caramyl phosphate
      Carbamyl phosphate + ornithine → citrulline + aspartate → fumarate + arginine → urea

    2. Glutamate + NH3 → glutamine (occurs in striated muscle and brain)

    3. Alpha ketoglutarate + NH3 → glutamate

      Serum NH3 is elevated in severe liver disease and in conditions like portal hypertension that cause shunting of blood from the liver.
      Potential cerebral toxicity of NH3; the active toxic form is free NH3, not the cation form, NH4. Evidence suggests that only free NH3 can freely diffuse across blood brain barrier. Because the PK of NH3 id 9.27 at 37 degrees C, small changes in blood pH cause large change in NH3 concentration; for example, with blood NH3 or 150.

      Blood pH

      Ratio NH4/NH3

      Conc. (NH3) mg%

      7.3

      94

      1.6

      7.4

      75

      2.0

      7.5

      59

      2.5

      7.6

      47

      3.1

      7.7

      37

      4.0

      7.8

      30

      4.8

  2. Bilibrubin

    1. "Direct-acting" – conjugated to one or two glucuronic acids

    2. Indirect (unconjugated) – tightly bound to albumin in serum. Serum albumin capacity is 60-80 mg bilirubin/100ml. Bilirubin displaced from albumin by salicylate, bile acids, sulfonamides.

    3. Source of bilirubin – primarily from hemoglobin, some from myoglobin, cytochromes, catalase and peroxidase; one gram hemoglobin yields 34 mg bilirubin; normally about 250 mg bilirubin produced daily; human can handle (excrete) about six times more than this per day.

    4. Urine bilirubin – only conjugated fraction (direct acting) in urine, unless there is renal disease.

    5. Urine urobilinogen – produced in gut by bacterial degradation of bilirubin, normally 1.5-3.5 mg/day. Ehrlich unit – color produced by 1 mg pure urobilinogen reacting with Ehrlich's aldehyde; elevated in hemolysis and some stages of hepatocellular disease. Low level in obstruction. Rarely, if ever, used now.

    6. "Delta bilirubin" – conjugated bilirubin covalently linked to albumin and with a t ½ similar to that of albumin. Is found in patients with longstanding jaundice.

8. Immunological Tests

  1. Hepatitis B surface antigen (HBsAg). The protein coat of the virus that causes hepatitis B. Found in serum in 100% of patients with hepatitis B but may be transient (present for only days). Also demonstrable in urine, tears and saliva of above patients. Present in patients with chronic hepatitis B.

  2. Anti-HBsAg (HbsAb). The antibody HBsAg. Implies exposure and immunity to hepatitis B.

  3. "e" antigen (HBeAg). A soluble antigen that is present only in individuals who also are positive for HBsAg and is a hepatitis B core protein.

  4. Anti-"e" (HBeAb). Also found in individuals positive for HBsAg. However, with antibody against "e," individuals are rarely infectious.

  5. Anti-HbcAg. Antibody to hepatitis B core proteins.. The hepatitis B core protein is manufactured in the hepatocyte nucleus and then released into the cytoplasm where it is coated with HBsAg to make the complete virus. AntiHBsAg is initially synthesized whole the B virus is actively replicating and thus, anti-HbcAg and HBsAg together are markers for continued infectivity. Anti-HBAg (HBcAb) and HBsAb together indicate recovery from hepatitis B and immunity against reinfection.

  6. Anti-A. Antibody to hepatitis A. IgG antibody implies previous exposure and immunity to hepatitis A; (infectious hepatitis). IgM antibody implies current hepatitis A infection.

  7. Anti-Delta. Antibody to delta hepatitis virus – may be IgM or IgG.

  8. Anti-C. Antibody present in sera of patients with chronic hepatitis C (formerly called non-A, non-B hepatitis). There are two ways to detect it. The Elisa test is sensitive but non-specific. If the Elisa test is positive, the specificity can be confirmed with a RIBA (radioimmunoblot assay), which is more specific.

  9. Autoantibodies

    1. Antimitochondrial antibody: an antibody that reacts with mitochondrial of all organs from many mammalian species. It is present in serum of 95% of patients with primary biliary cirrhosis and usually absent in patients with mechanical bile and duct obstruction. It is present in 1-5% of patients with other types of liver disease and in up to 15% of patients with chronic hepatitis. Its antigens are enzymes in the pyruvate dehydrogenase enzyme complex of mitochondria.

    2. Anti-smooth muscle antibody (SMA). Used by some in the diagnosis of chronic hepatitis but is of relatively little value. In some studies, its presence parallels the elevation of the aminotransferases; it is non-specific.

    3. Antinuclear antibody (ANA). Nonspecific antibody present in many patients with autoimmune chronic hepatitis and some with primary biliary cirrhosis.

9. Markers of Hepatic Synthetic Capacity

  1. Albumin (see above)

  2. Prothrombin time (PT): All of the major coagulation factors are synthesized in the liver (except for Factor VIII – made vascular endothelium and R-E cells). The PT is a measure of how long it takes blood to clot. It specifically assesses the extrinsic coagulation cascade (Factors I, II, V, VII, and X). In addition to liver dysfunction, vitamin K deficiency is another cause of a prolonged PT. Remember, Factors II, VII, IX, and X are vitamin K dependent. Therefore, in differentiating the cause for a prolonged PT, supplemental vitamin K is often administered and then the PT is rechecked. In pure vitamin K deficiency (from malnutrition for example) the PT should normalize after administration of vitamin K.

10. Other Techniques Used in the Diagnosis of Liver and Biliary Tree Disorders

  1. Liver scans

    1. Those dependent on uptake by R-E (reticulo-endothelial) cells

      1. Technetium bound to sulfur colloid

    2. Scans dependent upon uptake by parenchymal cells and excretion into bile – HIDA, PIPIDA, DISIDA and other iodine-containing chemicals.

    3. Uses of scan: Tc sulfur colloid and abscesses. HIDA-type scan are most useful to detect acute cholecystitis. They normally are concentrated in the liver and then the gallbladder. The cystic duct is usually occluded in acute cholecystitis and HIDA cannot enter the gallbladder. A positive HIDA scan is one in which the liver and common bile duct are seen but not the gallbladder.

  2. Oral and IV Cholangiography – rarely used today

  3. Percutaneous cholangiography

  4. Bile duct cannulation via Duodenoscopy (ERCP=endoscopic retrograde cholangiopancreatography)

  5. Ultrasonography

  6. CT Scans and MRI Scans