Liver disease: diagnostic evaluation (Proceedings)

The liver has more biochemical functions than any other organ of the body. It functions in hundreds of diverse metabolic activities including synthesis of plasma proteins; catabolism and storage of carbohydrates; synthesis, degradation, and storage of lipids; detoxification and excretion of many toxic agents; and the formation and elimination of bile. A variety of tests are available for detecting alterations in either hepatic structure or function. Proper selection of tests and knowledge regarding accurate interpretation (e.g. what level of increase in ALT activity is significant?) will enable the clinician to determine if there is significant disease of the liver and whether a liver biopsy is needed. Knowledge of diagnostic techniques and treatment principles for liver disease is important to small animal practitioners because of the prevalence of liver disease and the fact that many liver diseases are treatable if diagnosed early. In addition, increasing numbers of animal owners are seeking definitive medical care for their pets.

Diagnosis of liver disease

Diagnostic strategy

1. History

2. Physical examination

3. Hematologic studies

4. Biochemistries

5. Urinalysis

6. Serology

7. Radiography

8. Ultrasonography

9. Nuclear scintigraphy

10. Histopathology

Clinical signs associated with liver disease

• Depression

• Anorexia

• Diarrhea

• Fever

• Abdominal pain

• CNS signs

• Icterus

• Change in spleen size

• Dark brown urine

• Polyuria

• Pruritus

• Weakness

• Vomiting

• Weight loss

• Polydipsia

• Ascites

• Coma

• Hemorrhage

• Dark or light color stools

• Change in liver size

Clinical signs observed in hepatic encephalopathy

Physical examination

Physical examination findings in animals with liver disease range from minimal changes to more significant signs such as icterus, pigmented urine (bilirubinuria), bleeding diathesis (hematemesis, melena, hematuria, petecchiae, ecchymoses, pallor due to blood loss anemia), and hepatic encephalopathy. In some cases of acute hepatic disease, the liver is enlarged and painful (hepatodynia). Normally, the liver cannot be palpated in dogs and cats. Hepatomegaly is a feature of infiltrative disorders (inflammation, steatosis, neoplasia), congestion, or cholestasis (bile engorgement). Ascites may be present (due to hypoalbuminemia) and usually suggests chronic disease. Hemorrhage in the abdomen usually indicates trauma or neoplasia (e.g., hemangiosarcoma).

A thorough physical examination should always be done. In cats the earliest evidence of tissue jaundice is detected on the caudal hard palate. Extrahepatic physical findings may be helpful in suggesting possible liver involvement of a systemic process:

** The first step in evaluation of an icteric patient is to rule out hemolytic anemia (may be life-threatening). A PCV should be determined immediately in any compromised patient. Many dogs with icterus due to hemolytic anemia (e.g., AIHA) have been referred with a preliminary diagnosis of "liver disease".

Prehepatic causes (hepatic causes) of icterus include:

• Toxic causes

     o Methylene blue

     o Acetaminophen (cats)

     o Phenazopyridine (cats)

     o Lead

• Immune-mediated erythrocyte destruction

     o AIHA

     o FeLV-related (cats)

     o Transfusion reaction

• Infectious

     o Hemobartonella

     o Babesia

     o Ehrlichia

     o Cytauxzoon felis

     o Dirofilariasis (dogs)

Hematology

(to evaluate for leukemia, myeloproliferative disease, hemobart, characterization of anemia).

1. Inflammatory leukogram: Acute inflammatory diseases. Chronic suppurative diseases (cholangiohepatitis, liver abscess)

2. Mild nonregenerative anemia (normocytic/normochromic): Many chronic disorders

3. Mild microcytic anemia with leptocytes: R/O portosystemic shunt

4. Poikilocytosis: A nonspecific change in cats with any form of liver disease, including portosystemic shunt

5. Schistocytes (fragmented red cells): Hepatic neoplasms (especially HSA)

Biochemistries

The primary purpose of measurement of liver enzyme activity is to screen for the presence of hepatobiliary disorders. Serum enzymes lack specificity as to the nature or severity of the disorder. They are not liver function tests. Increased serum enzyme activity is quite common and may result from reversible or irreversible changes in the hepatocellular membranes, from structural injury associated with ischemia, necrosis, or cholestasis, or from microsomal enzyme induction (e.g., corticosteroids, phenobarbital). Microsomal enzyme induction is common in dogs but has not been recognized in cats. In most cases differentiation of the cause of increased liver enzyme values is not possible without a morphologic examination of liver tissue. In some cases of functional injury significant morphologic changes may not be present in hepatic tissue in cases in which liver enzymes were increased. Patterns of enzyme activity may support, but not prove, a definitive diagnosis. In some cases of severe liver disease (e.g., cirrhosis) there is an absence of increased enzyme activity.

1. ALT (SGPT)

     a. Located in abundance in the hepatocellular cytoplasm

     b. Found in several tissues, but greatest concentration is in hepatocytes (considered to be liver specific in dog and cat).

     c. Cytosolic: Concentration in cell is 10,000 times greater than in serum.

     d. ALT may leak from the hepatocyte in any condition that alters membrane permeability to a significant degree (leakage does not require cell death).

     e. Serum half-life <24 hours.

     f. Enzyme Increase

          (1) Magnitude directly proportional to number of hepatocytes affected.

          (2) After acute hepatocyte degeneration or necrosis, serum ALT levels rise by 12 hours. Highest elevations occur with cell necrosis.

          (3) Significant level: at least 2-3 times greater than normal. With severe necrosis may increase to 20 to 40 times normal.

          (4) Magnitude not related to reversibility of condition.

          (5) ALT elevations are not specific for primary hepatocellular disease. Bile duct obstruction (e.g., pancreatitis) causes ALT elevation within 3 to 4 days, and the elevation persists for at least several days. It is probable that retained bile salts physically damage the cell membranes of surrounding hepatocytes and result in enzyme leakage.

          (6) ALT is simply a screening test for hepatocellular damage.

          (7) Outcome:

               (a) If values decrease by 50% every 1-2 days, prognosis is good.

               (b) Continuing elevations suggest persistent hepatocellular disease (consider further tests: e.g., serum bile acids assay, liver biopsy).

2. AST (SGOT)

     a. NOT liver specific because of its high concentration in other tissues, especially muscle

                    (LDH has similar disadvantages).

     b. In cats, AST may be a more sensitive indicator of hepatobiliary disease than it is in the dog

     c. Tends to parallel ALT with respect to liver damage.

3. Alkaline Phosphatase (SAP)

     a. Increase is a result of increased production secondary to intrahepatic or extrahepatic cholestasis, not cell leakage as with ALT/AST.

     b. A membrane bound enzyme present in many tissues.

     c. Canine: 4 serum isoenzymes (bone, liver, corticosteroid induced [from the liver], isoenzyeme of unknown origin).

         Feline: 2 serum isoenzymes (bone, liver). Much shorter half-life than canine (any elevation must be evaluated critically). Smaller amount of AP per gram of liver.

     d. Increased SAP:

          (1) Osteoblast activity (2-3 times normal usually): Young growing animals up to 7-8 months (small increases in SAP which might be identified in a young growing cat due to bone isoenzyme may simulate the magnitude of increase of SAP in a young cat with liver disease. Bone tumors. Secondary renal hyperparathyroidism. FELINE HYPERTHYROIDISM is often associated with increased serum activity of hepatic enzymes (especially ALT and SAP). The AP isoenzyme may be of bone origin in these cases, owing to increased bone turnover secondary to the effects of thyrotoxicosis. Hyperthyroid cats do not have significant alteration of hepatic function in most cases.

          (2) Use of glucocorticoids (canine): Varies with the individual, type of glucocorticoid administered, and chronicity of treatment. If a liver biopsy reveals steroid hepatopathy, the R/Os include exogenous glucocorticoids, Cushing's Syndrome,increased endogenous glucocorticoids possibly related to chronic stress/illness.

          (3) SAP (hepatic) synthesis induced by drugs other than glucocorticoids (e.g., anticonvulsants).

          (4) Primary or secondary hepatocellular disorders. Largest SAP increases occur with diffuse or focal cholestatic disorders and hepatic neoplasms.

          (5) Any cause of hepatobiliary inflammation may result in secondary hepatobiliary disturbances leading to intrahepatic cholestasis. **Pancreatitis may result in marked increases in SAP.

          (6) Mammary neoplasia (mild).

     e. Typically there is a lag period between liver changes and reflection of those changes in SAP activity

4. Gamma Glutyl Transferase (GGT)

     a. Most serum GGT activity in the dog and cat is derived from liver. Largest tissue activity is in kidney and pancreas.

     b. Increased GGT:

          (1) Intrahepatic or extrahepatic cholestasis

          (2) Pancreatitis

          (3) Drug induction (canine).

     c. In canine, increased GGT largely parallels increased SAP. Exception - there is a minimal induction of GGT activity with anticonvulsant medications (may be useful in differentiating primary liver disease from changes in enzymes caused simply by drug induction).

     d. In feline, GGT is more sensitive in diagnosis of liver disease than is alkaline phosphatase (but is more specific for hepatobiliary disease). GGT generally parallels changes in SAP with one notable exception in cats. Cats with hepatic lipidosis have marked increases in SAP while only mild increases in GGT occur. This fact may be useful for strengthening suspicion that hepatic lipidosis is present.

5. Nonspecific elevations of serum hepatic enzyme activity can occur secondary to a number of causes. In most cases serum enzyme values do not increase beyond 2 or 3 times normal. Usually there is an absence of substantial morphologic or functional hepatobiliary injury.

     a. Disorders causing decreased hepatic perfusion with subsequent hepatocellular hypoxia: shock, cardiac disease (CHF, heartworm disease, pericardial effusion), anemia.

     b. Endotoxemia/septicemia

     c. Diabetes mellitus

     e. Hypothyroidism

     f. Hyperthyroidism

     g. Hyperthermia

6. Bilirubin

     a. The total bilirubin level provides the most useful clinical information (there is little value in studying any quantitative differences between direct and indirect bilirubin levels).

     b. Classify jaundice as: Prehepatic, Hepatic, Posthepatic. Jaundice develops earlier in the disease process than with parenchymal disease.

     c. Jaundice develops when bilirubin levels exceed 2.0 mg/dl to 3.0 mg/dl.

     d. Bilirubinuria, even in concentrated urine, is an abnormal finding in cats. Cats have a high renal threshold for bilirubin, while dogs have a low renal threshold.

     e. Bilirubin is less sensitive but more specific than liver enzymes in identifying hepatobiliary disease. Using bilirubin values in conjunction with liver enzymes and serum bile acids concentrations improves the diagnostic specificity of the enzymes.

7. Plasma Proteins

     a. Albumin is the major plasma protein synthesized in the liver.

          (1) Albumin half-life is approximately 23 days in the dog.

          (2) Decreased albumin levels from synthetic failure ensues when greater than 70% of hepatobiliary function has been compromised.

     b. Most of the serum globulins, with the exception of immunoglobulins, are synthesized in the liver.

          (1) Patients with hepatic failure rarely have clinically significant decreased globulin levels; however globulin concentrations are often normal or even increased in hepatobiliary disease due to increases in systemic immunoglobulin synthesis (lymph nodes, bone marrow, spleen, other organs) in response to systemic antigenic challenge.

     c. Problem - Hypoproteinemia

IF HYPOPROTEINEMIA IS IDENTIFIED, NEXT INVESTIGATIVE STEP:

          (1) Liver: bile acids assay.

          (2) Kidney: urine protein:creatinine ratio

          (3) Intestine: biopsy.

          Definitive diagnosis is then made by obtaining biopsies from the involved organ(s).

Serum bile acids - basis of the test

Abnormal serum bile acid concentration approaches 100 percent specificity for hepatic disease when concentrations exceed 20 uM/L in cats (normal 5 uM/L)and 30 uM/L in dogs (normal 10 uM/L). The primary bile acids, cholic and chenodeoxycholic acids, are synthesized in the liver from cholesterol and then conjugated to an amino acid (primarily taurine in dogs and cats). Following synthesis the bile acids are stored and concentrated in the gallbladder. Cholecystochinin is released in the small intestine in response to the presence of hydrochloric acid, dietary fats, and proteins and it stimulates contraction of the gallbladder with subsequent release of bile acids. In the small intestine bile acids facilitate fat emulsification, digestion, and absorption. Most of the primary and secondary bile acids are reabsorbed in the ileum by an efficient active transport process and are rapidly extracted from the portal circulation by the liver.

The enterohepatic bile acid circulation is extremely efficient (90 percent). An estimated 2-5 percent of the total circulating bile acid pool is lost in the feces each day. The efficiency of this enterohepatic circulation is the basis for the use of bile acid concentrations in the assessment of hepatobiliary function and of the integrity of the hepatoportal circulation The liver maintains a tremendous reserve capacity for bile acid synthesis. Hepatic failure does not limit serum bile acid concentration.

It is not necessary to run a bile acid assay in a hyperbilirubinemic patient that is not anemic. Hyperbilirubinemia in the absence of hemolysis indicates that a hepatic or post hepatic disorder is present. However, the test may be run in hyperbilirubinemic patients because bile acids do not compete with bilirubin for transport or metabolism as do the organic anion dyes (BSP and ICG). The degree of bile acid elevation in icteric cats is greatest with hepatic lipidosis and extrahepatic bile duct obstruction and in dogs with severe liver disease

Performance of test

To maximize the diagnostic information from total serum bile acid measurement, both a 12 hour fasting and a 2 hour postprandial sample should be obtained. An exception is that if bile acid evaluation is done in a jaundiced cat with a total bilirubin level of greater than 3.0 g/dl only a resting level is done. Foods that are high in protein and fat should be fed because they most consistently challenge the bile acid enterohepatic circulation at the 2 hour postprandial level. It is not necessary to feed a large amount of food. Animals 10 pounds or less are fed 2 teaspoons, and those greater than 10 pounds 1 to 2 tablespoons.

If inappetence is a problem, the following steps can be taken to coax a cat to eat:

1. Force feed.

2. Special steps to avoid anorexigenic stimuli may be necessary (e.g., offer food in a quiet environment - away from dogs [!!], have the owner hand feed in a private area, etc.). In addition, mild stimulation by petting and stroking the back may stimulate eating.

3. Warming the food may help coax an inappetent cat to eat. Cats prefer food served at 78 to 103 degrees F.

4. Administer diazepam IV to stimulate the appetite (0.1 mg/lb).

5. If the animal's refusal to eat is thought to be primarily due to being in the hospital environment and force-feeding and other measures prove unsuccessful the fasting blood sample is drawn and the patient is sent home to eat. The postprandial blood sample is then obtained at 2 hours.

In animals with encephalopathic tendencies, where there is concern about feeding high protein foods, a low protein meal (e.g., Hill's k/d, Select Care Modified) can be fed. If vomiting is a problem and its frequency precludes feeding, initial testing is limited to a fasting sample. If the result is above normal then a significant hepatic problem remains a consideration. However, if the fasting sample is normal liver disease cannot be ruled out.

Interpretation

Fasting or postprandial values exceeding 20 uM/L in cats and 30 uM/L in dogs are considered to indicate the presence of significant hepatobiliary disease. A liver biopsy is warranted in these patients. The pattern of postprandial increase from fasting values can be diagnostically significant. Some patients with severe impairment of hepatobiliary function have normal fasting bile acid levels but profound elevations after feeding. Animals with severe cholestasis generally have high fasting serum bile acid concentrations that are relatively unchanged or increase only slightly after a challenge meal. Cats and dogs with portosystemic shunting may have normal or only slightly increased fasting levels, but postprandial concentrations are always abnormal.

Bile acid levels can be very useful for monitoring response to therapy after a definitive diagnosis has been obtained. The SBA concentration is a much more sensitive indicator of hepatic function than serum bilirubin or albumin. Liver enzymes will often return to normal long before liver function returns to normal.

Radiography and ultrasonography

Survey radiography of the abdomen may produce useful information in animals with suspected hepatobiliary disease. Cats with liver disease frequently have normal liver size to slight to moderate enlargement. An abnormally small liver (microhepatica) is most commonly found in conjunction with congenital portosystemic shunt or chronic severe liver disease (e.g., cirrhosis). Radiographic signs of microhepatica include cranial shifting of the stomach axis, and in some cases cranial displacement of the kidneys and intestines. Radiographic signs of hepatomegaly include extension of the liver margins caudal to the costal arch, rounding of the liver borders, and caudal displacement of the stomach, kidneys, and intestines. Large focal masses are sometimes visualized on survey radiographs. Other abnormalities that may be identified include choleliths (increased opacities in the region of the gallbladder), calcified intrahepatic lesions, or focal or multifocal radiolucencies that may indicate presence of hepatobiliary infection, abscessation, or cholecystitis.

Contrast portography is used to establish definitive diagnosis of a portosystemic shunt. Ultrasonography is useful for detecting the presence of a shunt in some cases but localization may be difficult. Rectal portal scintigraphy is an excellent technique for noninvasively confirming the presence of a shunt, but this technique does not reliably identify specific location of the shunt. Contrast portography establishes both presence and position of the shunt vessel.

Ultrasonography generally provides more useful information than survey radiography in noninvasively evaluating liver parenchyma and the biliary system. It is an invaluable diagnostic aid for evaluating patients with jaundice. Focal or diffuse changes in parenchymal echogenicity (hypoechoic, hyperechoic), bile ducts, gallbladder size and content, and vasculature may be evaluated with ultrasonography. Focal hepatic lesions may be readily identified and subsequently sampled via fine needle aspiration or needle biopsy under ultrasound guidance. The gallbladder is readily imaged, but the rest of the biliary tract is not identified in the normal animal. Dilatation of the biliary tract occurs in a retrograde manner from the site of obstruction with rapid enlargement of the gallbladder Dilatation of the extrahepatic ducts sufficient for ultrasonic detection occurs in about 72 hours. Intrahepatic biliary duct enlargement is seen after approximately five to seven days. If dilatation of the extrahepatic ducts is not observed on ultrasound examination in a jaundiced cat, and hemolytic disease has been ruled out, hepatocellular disease is most likely present. A liver biopsy is warranted to determine a definitive diagnosis.

Ultrasonography can provide very useful information in provisional differentiation of hepatic lipidosis from other major causes of feline liver disease, including extrahepatic bile duct obstruction, cholangiohepatitis, and metastatic neoplasia. Ultrasonographic features of hepatic lipidosis include diffuse homogeneous hyperechogenicity of the hepatic parenchyma and hepatomegaly. In hepatic lipidosis, liver echogenicity is equivalent to or greater than that of spleen and renal cortex (in normal animals the liver is less echogenic than the spleen and equivalent or slightly more echogenic than the renal cortex).

Liver biopsy

The most important diagnostic step in definitive diagnosis of liver disease is histologic evaluation of liver tissue. The diagnostic tests that have already been described are very important for determining the likelihood of a hepatic disorder as well as providing information about liver function and whether or not a surgical approach is indicated for definitive diagnosis and/or management. Diagnosis, specific decisions regarding treatment, and discussion regarding overall prognosis all depend on liver biopsy findings, as well as on biopsy of other organs as indicated (i.e., there may be a multifocal disease process). There are many acceptable methods of obtaining liver biopsies. There are very few contraindications to doing the procedure. Liver biopsy procedures include fine-needle aspiration, percutaneous blind or ultrasound-guided needle biopsy, laparoscopy-guided, and exploratory laparotomy.

Wedge biopsies obtained either at laparoscopy or laparotomy are strongly recommended, as the larger wedge samples provide more tissue for analysis. Liver biopsy tests for dogs should always include histopathology, copper quantitation, and aerobic and anaerobic cultures. Tests for cats should include histopathology and aerobic and anaerobic cultures.