Chapter 8 The Clinical Pathological Assessment of the liver

 

 

                     

  Introduction:

The history and findings of the physical examination for animals with hepatic disease are often no-specific. When clinical signs are seen, they are often multisystemic signs that are not specific to the liver. The history usually includes recurrent abdominal disorders such as anorexia, vomiting, and diarrhea, and sometimes includes vague central nervous system signs of depression and lethargy. Findings on physical examination mat include a palpably enlarged liver, ascites, and clay colored feces. Jaundice, in the absence of anemia, is a good indication of hepatic disease. Evidence of pain on palpation of the liver results from pressure on the hepatic capsule and indicates acute disease. Chronic hepatic disease rarely causes a painfully distended capsule.

The liver is a complex organ that performs many metabolic functions which are dependent upon the integrity and interaction of four anatomic subunits:

• (1) the hepatic parenchymal cells,
• (2) the biliary system,
• (3) the hepatic vascular system, and
• (4) the Kupffer or reticuloendothelial system.

Because of this complexity, no single test of liver function is sufficient for clinical assessment of most liver problems. It is usually necessary to use a battery of tests or a profile of several liver function tests to define the problem.

8.1.1 General classification of hepatopathies

1. Acute

• a. Non-obstructive
• b. Obstructive

2. Chronic

• a. Non-obstructive
• b. Obstructive 

8.1.2 Basic results of hepatopathies

Hepatic disease most often involves a combination of the parenchymal cells, bile ducts, and/or vascular system and is manifested as follows:

1. Leakage of hepatocellular substances into the ECF

• a. Due to reversible damage or necrosis of the hepatic cells.
• b. Assays measure predominantly endogenous substances, e.g., serum enzyme activity.

2. Reduction of functional hepatic mass

• a. Due to conditions such as hepatic atrophy, fibrosis and neoplasm.
• b. Assays involve predominantly the administration of exogenous substances such as dyes or diets.

3. Biliary obstruction and/or cholestasis

• a. Assays involve both endogenous and exogenous substances.

8.1.3 Methods of Assay

There are three types of tests for evaluating hepatic disease:

• 1. Those that measure serum enzyme activity. The mechanisms of serum enzyme alteration include:
  • a. Alteration in cell membrane permeability
    • 1) Inflammatory reactions
    • 2) Cellular degeneration
    • 3) Increased cellular activity
    • 4) Fatty metamorphosis
  • b. Cell necrosis
  • c. Impaired clearance of enzymes from serum
  • d. Impaired synthesis of enzymes
  • e. Increased production of enzymes
• 2. Those that measure bilirubin or organic dye secretion and excretion.
• 3. Those that measure a specific metabolic function such as plasma proteins. 

8.2 Biochemical Indicators of Hepatocellular Damage

8.2.1 Introduction:

Evaluation of non-plasma specific enzyme levels, i.e., enzymes with no known physiological function in plasma, is a valuable non-invasive technique for determining hepatocellular integrity. Two subclasses of these enzymes exist,

• (1)Enzymes associated with cellular metabolism and
• (2) Enzymes of secretion. Enzymes of the first class can be used as indicators of hepatocellular membrane leakage or necrosis.

To be useful, an enzyme indicator must meet the following criteria:

• 1. It must be as specific as possible for the organ being evaluated.
• 2. Its levels must become measurably elevated in serum following damage to the organ.
• 3. The normal serum range for the enzyme must be well-defined and reasonably narrow with a minimal overlap between normal and abnormal values.
• 4. An efficient method of assay must be available and should be as specific and convenient as possible. In general, these methods involve:
  • a. Measurement of the disappearance of substrate or change in substrate concentration.
  • b. Measurement of an end-product.
  • c. Measurement of change in amount of a coenzyme or cofactor at a specified time, with the rate of change being the measure of enzyme activity.

Few enzymes fulfill all of the above criteria, however, a few are considered "liver specific" in that high concentrations are present primarily in liver tissue. Other more nonspecific enzymes may be used in the evaluation of the liver if other organs are known to be free of pathology. A comparison of the levels of several different enzymes, i.e., enzyme profiles, may be used to evaluate liver disease.

8.2.2 General Remarks:

• 1. Alteration of hepatocyte plasma membrane permeability is the only mechanism predisposing to leakage and may be due to:
  • a. decreased O2 supply to hepatocytes (anoxia)
  • b. direct effect of certain toxins
  • c. inflammation
  • d. metabolic disorders
• 2. Increased serum enzyme levels are signalment of hepatocellular disease:
  • a. The rate of enzyme leakage is dependent upon
    • 1) the extent, severity and rapidity of lesion
    • 2) the concentration, rate of synthesis, subcellular localization, size and diffusibility of the enzyme
  • b. The magnitude of increase is directly proportional to the number of hepatocytes affected.
  • c. The magnitude of increase is not related to the reversibility of the lesion.
• 3. The type of enzyme elevation present and the duration of the elevation may be of prognostic value.
  • a. The presence of absence or ratio of cytosol and mitochondrial elevation may be of prognostic value.
    • 1) Generally, the more severe the lesion, the more likely the presence of mitochondrial leakage.
  • b. The persistence of enzyme markers of severe necrosis in the serum indicates a poor prognosis.
  • c. Falling levels of enzyme markers of hepatocellular damage after several days indicates a good prognosis.
• 4. The duration of enzyme increase in the serum is dependent on persistence of leakage from the hepatocytes and the rate of clearance from plasma.
  • a. The enzymes are not excreted but are stereochemically denatured.
  • b. The plasma half-life of most enzymes varies from a few hours to several days.

8.2.3 The Enzymes

1. Sorbital Dehydrogenase (SDH, SD, Inositol Dehydrogenase)

• a. Function: 

• 1) Sorbital is a polyhydric alcohol derived from glucose
• 2) Conversion takes place mainly in the liver

• b. Source:
  • 1) Liver
    • a) contains largest amounts of SDH
    • b) enzyme is located in the cytosol of the hepatocytes
    • c) highly concentrated in the livers of horses, rats, dogs, cattle, cats, sheep, rabbits, mice and pigs
  • 2) Kidney
    • a) in the pig, contains approximately 50% as much SDH as the liver
    • b) lesser amounts in other species
  • 3) Brain
  • 4) Small Intestine
    • a) in the horse, contains more than the kidney but less than the liver
  • 5) Skeletal Muscle and RBC
    • a) minimal amounts of SDH
    • b) no increase in serum SDH following IM injection as seen with AST and CPK
• c. Interpretation:
  • 1) Marked increase in serum levels
    • a) liver necrosis - if one insult, levels will return to normal within 24 hours.
  • 2) Moderate increase in serum levels
    • a) pancreatitis
    • b) cirrhosis
    • c) obstructive jaundice
    • d) Diabetes mellitus
    • e) acute intestinal obstruction (horse)
    • f) acute enteritis (horse)
    • g) grass sickness (horse)
  • 3) Mild increase in serum levels
    • a) severe azoturia - after a few days
    • b) influenza
    • c) arteritis
    • d) prolonged corticosteroid administration

NOTE: Hepatic injury appears to be the only source for significant SDH activity elevation in serum.

• d. Collection and Assay:
  • 1) The enzyme is very unstable. After 24 hours at room temperature, the serum has 1.4% of its original activity. After 24 hours at -25°C, the serum has 88% of its original activity.
  • 2) The assay should be run within 24 hours, preferably 12 hours, of collection.
  • 3) A serum clot tube is the sample of choice, however, a heparinized plasma sample may be used.
  • 4) EDTA, oxalate, and other metal chelating anti- coagulants decrease SDH activity.
  • 5) Most commercially available assays involve the conversion of fructose to sorbitol and measure the rate of decrease in absorbance at 340 nm that results from the oxidation of the cofactor NADH.

2. Alanine Aminotransferase (ALT, SGPT, SALT, Glutamic Pyruvic Transaminase)

• a. Function: 

• 1) ALT is involved in enzymatic transamination, the transfer of an amino group from an amino acid to a keto acid.
• 2) This process is necessary for the excretion of the amino group.

• b. Source
  • 1) Liver
    • a) High ALT concentrations have been observed only in the hepatic parenchymal cells of dogs, cats, rats and primates.
    • b) The livers of other domestic species do not contain a significant level of ALT.
    • c) The enzyme is located in the cytosol and mitochondria of the hepatocyte with the higher concentration being in the cytosol of most species.
  • 2) Kidney, heart, skeletal muscle, pancreas, spleen, lung, and RBC
    • a) Levels are not significant in small animals, however in large animals, levels in cardiac and skeletal muscle may exceed those of the liver.
    • b) Kidney levels in small animals exceed those of the other tissues but are much less than that found in the liver.
• c. Interpretation:
  • 1) Marked increase (e.g., >400 IU/L) in serum levels occurs in severe liver necrosis, infectious and toxic hepatitis.
  • 2) Moderate increase in serum levels occurs in minor to moderate necrosis, obstructive jaundice, metastatic carcinoma and hepatic congestion.
  • 3) Mild increase in serum levels occurs with slight degenerative changes, fatty change and with cholestasis.
  • 4) After hepatic necrosis and regeneration, levels of ALT may remain elevated from one to three weeks.
• d. Collection and Assay:
  • 1) The stability of the enzyme is questionable
    • a) There appears to be some species variation.
    • b) Serum stored at room temperature should be assayed within 8 hours.
    • c) Serum stored at refrigerated temperatures should be run within 3 days.
    • d) Serum stored at -70°C varies in stability with species.
  • 2) A serum clot tube is the sample of choice, however, heparinized plasma sample may be used.
  • 3) Lipemia and hemolysis greatly interfere with several assay methods.
  • 4) Significant hemolysis should also be avoided due to ALT activity in erythrocytes.
  • 5) The more accurate assay methodologies involve enzyme kinetics, usually in a two step process.

3. Aspartate Aminotransferase (AST, SGOT, SAST, Glutamic Oxaloacetic Transaminase)

• a. Function: 

• 1) AST catalyzes the transfer of an alpha-amino group from aspartic acid to a keto acid (usually alpha-ketoglutaric acid).
• 2) This process is necessary for the excretion of the amino group.
• 3) Oxaloacetic acid is an important component of the Tricarboxylic acid cycle.

• b. Source:
  • 1) AST is located in both the cytosol and mitochondrial fractions of the cell. The enzyme differs markedly in each location (isoenzymes).
  • 2) AST is located in almost all tissues and in all domestic species of animals.
  • 3) The highest concentrations are located in:
    • a) skeletal muscle cells - highest concentrations
    • b) liver
    • c) heart muscle
  • 4) Smaller amounts are located in:
    • a) kidney
    • b) pancreas
    • c) brain
    • d) spleen
    • e) RBC
• c. Interpretation:
  • 1) Since all major tissues contain high concentrations of AST, increased AST levels are not definitively indicative of hepatic necrosis unless diseases of other large organ systems can be ruled out.
  • 2) The upper limits of normal AST activity in the horse is considerably greater than that observed in other species.
  • 3) Increased levels indicate:
    • a) Skeletal muscle source dominates serum levels
      • (1) exercise can cause increased serum levels of up to 150 IU/L
      • (2) muscular damage (e.g., azoturia, "Tying-up" syndrome) can cause significant increase in serum levels of 200-2000 IU/L
    • b) liver damage (increases from 200-1000 IU/L)
    • c) Myocardial infarction
    • d) intestinal complications
    • e) septicemia
  • 4) Certain drugs can also elevate AST
    • a) salicylates
    • b) corticosteroids
    • c) estrogens
    • d) androgens
    • e) antibiotics - erythromycin, lincomycin, gentamycin
    • f) phenothiazine
    • g) anesthetic agents - halothane
• d. Collection and Assay
  • 1) Enzyme stability
    • a) room temperature for 3 days
    • b) refrigerated for 1 week
    • c) frozen (-25°C) for 1 month
    • d) frozen (-70°C) for 1 month
  • 2) A serum clot tube is the sample of choice, however, a heparinized plasma sample may be used.
  • 3) Lipemia and hemolysis greatly interfere with several assay methods.
  • 4) The more accurate assay methodologies involve enzyme kinetics, usually in a two step process.

4. Serum Arginase

• a. Function 

• 1) Arginase functions in the urea cycle which is entirely within the liver.

• b. Sources:
  • 1) Liver
    • a) Arginase is considered to be a liver specific enzyme because considerably higher concentrations have been found in hepatocytes than in any other tissue.
    • b) At first thought to be a mitochondrial enzyme, arginase is now known to be localized in the cytosol.
    • c) It is present in all ureotelic (urea excreting) animals.
  • 2) Arginase isoenzymes have been reported in the heart, epidermis, submaxillary gland, kidney and brain.
• c. Interpretation:
  • 1) Greatly increased serum activity indicates a necrotic process in the liver.
    • a) There is a rapid disappearance of the enzyme from the serum.
      • (1) If both arginase and ALT are continuously elevated in the serum, the indication is a progressive hepatic necrosis.
      • (2) If ALT is elevated but arginase has returned to normal levels, the prognosis is more favorable.
• d. Collection and Assay
  • 1) Arginase is relatively unstable at room temperature.
    • a) If the assay cannot be run within 4 hours, the sample should be refrigerated.
    • b) Arginase in serum is stable for 7 days at refrigerated temperatures.
  • 2) A serum clot tube is the sample of choice.
  • 3) Lipemia and hemolysis affect the results and such samples should not be used.
  • 4) The most widely used methodology involves assessment of the amount of product (ornithine) formed. No commercial method is available at this time.

5. Lactate Dehydrogenase (LDH)

• a. Function: 

• 1) LDH catalyzes the reversible oxidation of L-Lactate to pyruvate. 

• b. Source:
  • 1) LDH is located in body tissues that utilize glucose for energy. It is a cytosolic enzyme.
  • 2) The activity of LDH in the RBC is 150x the activity in plasma; thus, hemolysis alters plasma LDH activity.
  • 3) LDH is a tetrapeptide made up of two peptides, H (heart) and M (muscle). These combine to make 5 isoenzymes: LDH1 (H4), LDH2 (H3M1), LDH3 (H2M2), LDH4 (M3H1), and LDH5 (M4).
  • a) LDH1 and LDH2 are located in heart, RBC, kidney, brain and liver (cow and sheep)
  • b) LDH3 is located in lung, pancreas, adrenals, spleen, thymus, thyroid, lymph nodes and leukocytes
  • c) LDH4 and LDH5 are located in myocardium, skeletal muscle, brain and liver (in most species)
• c. Interpretation:
  • 1) Specific isoenzyme profiles may occur in hepatic disease, specific malignancies, muscular damage and nephrotoxicosis. These profiles vary greatly with species.
  • 2) LDH is more widely used in human medicine.
  • 3) LDH may be elevated in any process in which there is cell necrosis.
• d. Collection and Methods:
  • 1) Stability
    • a) Room temperature for 1 week
    • b) Refrigerated for 1-3 days
    • c) Frozen for 1-3 days (freezing may alter isoenzyme pattern)
  • 2) Serum clot tube preferred but heparinized plasma may be used.
  • 3) EDTA, oxalate, etc., indirectly inhibit the enzyme.
  • 4) Hemolyzed samples should not be used.

6. Glutamate Dehydrogenase (GD, GLDH) 

• a. Function:

• 1) Excess amino acids cannot be stored or excreted by the body so they are converted into metabolic intermediates by removal of an amino group. Once transferred to glutamate by the transaminases, the amino group is then removed by GD.
• 2) Alpha-ketoglutarate is an important component of the citric acid cycle. 

• b. Source:
  • 1) GD is quite liver specific, located mainly in the mitochondria.
  • 2) Other organs such as muscle contain some but do not appear to influence serum levels.
• c. Interpretation:
  • 1) Liver Disease
    • a) Increased levels in serum may indicate more severe cellular lesions than increases in SDH or AST.
    • b) GD is probably the enzyme of choice for cattle and is more sensitive and specific than AST.
• d. Collection and Methods:
  • 1) Serum clot tube is the sample of choice.
  • 2) Commercial kits for the assay are no longer available.

7. Isocitrate Dehydrogenase (ICD)

• a. Function: 

• 1) ICD is involved in the citric acid cycle.

• b. Source:
  • 1) The citric acid cycle takes place in the mitochondria; thus, the enzyme ICD is located mainly within the mitochondria.
  • 2) ICD is not organ specific and has a distribution similar to AST (predominantly liver, kidney, heart and skeletal muscle).
• c. Interpretation:
  • 1) Increased levels of ICD may indicate liver necrosis if other system abnormalities are ruled out.
• d. Collection and Methods:
  • 1) Sigma method is available which involved a kinetic assay.

8. Ornithine Carbamyltransferase (OCT)

• a. Function: 

• 1) OCT functions in the urea cycle.

• b. Source:
  • 1) The urea cycle is located only within the liver in all urea secreting animals.
  • 2) The first steps of the urea cycle take place within the mitochondria, the latter steps within the cytosol.
  • 3) Therefore, OCT is liver specific and mainly mitochondrial in location.
• c. Interpretation:
  • 1) Elevated levels indicate liver necrosis. 
• d. Collection and Methods:
  • 1) the assay method is cumbersome and requires several hours for results. 

8.3 Biochemical Indicators of Hepatic Obstruction or Cholestasis

8.3.1 Causes of Cholestasis:

• 1. Decrease in bile volume from altered bile salt concentration (affects hepatic membranes).
• 2. Interference of drugs and abnormal bile acids with normal biliary micelle membranes.
• 3. Interference with sodium and water transport into the bile.
• 4. Increased intraluminal biliary pressure due to obstruction by:
  • a. Hepatocellular swelling
  • b. Inflammation
  • c. Neoplasia
  • d. Biliary atresia
  • e. Calculi

8.3.2 Serum Enzymes

1. Alkaline Phosphatase (AP, ALP)

• a. Function:
  • 1) AP are zinc metal enzymes of low substrate specificity which catalyze hydrolysis of monophosphate esters (glucose, glycerol, etc.) at an alkaline pH (e.g., 9.0).
• b. Source:
  • 1) The highest activity for AP is in the microvilli of secretory and absorptive cells.
    • a) Osteoblasts and chondroblasts
    • b) Hepatobiliary system - hepatocytes and biliary epithelium
    • c) GI mucosa
    • d) Renal tubules
    • e) Placenta
    • f) Leukocytes
    • g) Mammary gland
  • 2) AP is a microsomal enzyme in most cells.
  • 3) There are several major isoenzymes of AP.
    • a) In the young animal, the major isoenzyme of AP is from bone.
    • b) In the mature animal, the major isoenzyme of AP is from the liver. The liver has several internal isoenzymes which vary depending upon the hepatopathy present.
• c. Interpretation:<