Clinical Case Presentation

Sofia, an 8 y.o. female spayed asymptomatic Labrador retriever presented with an increase in ALT (discussion on canine idiopathic chronic hepatitis in the dog, including canine copper-associated hepatitis).

Interpretation of complete blood count (CBC), biochemistry profile and liver function assays in hepatobiliary disease in the dog.

CBC

• Poikilocytosis: including acanthocytes, echinocytes, target cells, and stomatocytes (reflecting a decreased tolerance to oxidative stress)
• Anemia of chronic disease or chronic GI blood loss
• Microcytic and hypochromic anemia (iron deficiency)

Biochemistry Profile

I- Markers of Hepatocellular Injury

Increases in ALT (Alanine Aminotransferase)

• Fairly liver specific: although also found in liver, muscle (cardiac and skeletal) and kidneys
• Increases occur due to cell damage (increased membrane permeability or necrosis) and induction (increased synthesis).
• Serum half-life is about 40–60 hours in the dogs: following an acute hepatic injury, ALT will increase within 12 hours and serum enzyme activity generally peaks at about 24–48 hours and then begins to decrease.¹
• Generally considered significative if increase is 2x normal.
• In healthy dogs, the critical change value for ALT was found to be 47.7%.² In other words, ALT activity must change by at least 47.7% for it to be considered statistically.
• The degree of elevation of serum ALT activity is roughly proportional to disease severity and affected hepatic mass. However, liver injury can be present despite a normal ALT activity due to a decrease in the number of hepatocytes (i.e., advanced fibrosis, portosystemic shunting), in cases of non-inflammatory primary or secondary neoplasia (e.g., hepatocellular carcinoma or hemangiosarcoma), and potentially very early in the course of disease.
• Increased ALT supports hepatocellular injury, but does not provide information on liver function.

Generally speaking

• Mild increases (up to 2-fold) can be rechecked every 2 weeks and possibly treated with nutraceutical hepatoprotectants. If increase persists more than 4–6 weeks, further investigation is warranted.
• Moderate increases (2- to 5-fold increases) require a diagnostic workup (e.g., liver function assays [bile acids or ammonia] and leptospirosis serology/PCR).
• Severe increases (5-fold increases) or persistent increases in ALT warrant an immediate diagnostic workup, likely including liver biopsies.

Table 1. Causes of increased serum ALT activities in dogs

Primary Hepatopathies

Inflammatory (ex: acute hepatitis, idiopathic chronic hepatitis including copper-associated chronic hepatitis, lobular dissecting hepatitis, gallbladder mucocele)
Neoplasia (primary: hepatocellular carcinoma, lymphoma; metastatic)
Infectious (leptospirosis, infectious canine hepatitis, toxoplasmosis, Heterobilharzia infection)
Trauma (contusions, herniation, lobe torsion)
Nodular hyperplasia

Secondary Hepatopathies

Hyperadrenocorticism
Diabetes mellitus

Local or systemic inflammation (enteritis, pancreatitis, peritonitis, SIRS, septicemia, anaphylaxis)
Cellular hypoxia (anemia, congestive heart failure, thromboembolism, circulatory shock)
Metabolic (storage diseases)

Toxic

Drugs (ex: glucocorticoids, barbiturates, carprofen, antimicrobials (ex: TMS, tetracycline), azathioprine, ketoconazole)
Toxin ingestion (aflatoxin, Amanita mushroom, blue-green algae, copper, herbicides/insecticides, iron, sago palm, zinc, xylitol)

Extra-hepatic Sources of ALT

Severe muscular injury (uncommon; higher increases in AST are typically seen)

Increases in AST (aspartate aminotransferase):

• Not specific to the liver (present in muscles, but also in brain, liver, kidney and erythrocytes)
• Muscle damage (CK activity may help differentiate origin as it should remain normal unless there is concomitant muscle disease) and hemolysis can cause considerable increases in AST activity
• The enzyme half-life is about 22 hours in the dog.

II- Markers of Cholestasis

Increases in ALP Activity (Alkaline Phosphatase)

• Can indicate primary hepatobiliary disease, such as cholestasis, as well as canalicular cell necrosis, or alternatively increased hepatic synthesis.
• Not specific to the liver, as several ALP isoenzymes have been identified in liver, bone, intestines, kidney and placenta.
• An increase in ALP has a good sensitivity (86%), but a poor specificity (49–51%) for the diagnostic of hepatobiliary disease.
• The specificity of ALP for the diagnosis of hepatobiliary disease increases to 94% if combined with an increased serum GGT.³
• Considered significant if increase is 2–3x normal.
• The enzyme half-life is about 72 hours in the dog.
• Enzymatic induction (isoenzyme):
• Hepatic: with usage of glucocorticoids or anticonvulsant therapy in dogs (ex.: phenobarbital)
• Bone: in young animal or with bone tumor

Increases in GGT Activity (Gamma-glutamyltransferase)

• Increases parallel ALP
• In dogs, GGT is often considered more specific but less sensitive than ALP for the detection of hepatobiliary disease.
• GGT has a half-life of approximately 72 hours in dogs.⁴

Hyperbilirubinemia

• Poor sensitivity and specificity for hepatobiliary disease
• May be caused by hemolysis, primary hepatic disease and extrahepatic cholestasis

Important: Changes on biochemistry profiles suggestive of liver dysfunction/failure include hypoalbuminemia, decreased urea, hypoglycemia (liver failure) and hypocholesterolemia.

Liver Function Assays

Ammonia (fasted):

• Degradation product of proteins in the GI tract ®portal circulation ®urea cycle (amino acids synthesis)
• Relatively insensitive marker of hepatic function, and it has been suggested that >70% reduction of hepatic function is required for serum ammonia concentration to be increased⁵
• The sensitivity of plasma ammonia for the detection of congenital portosystemic shunts has been reported to vary between 81–100% in dogs.^5,6
• Needs to be analysed within the hour (volatile; heparinized sample on ice)

Bile acids:

• Normal cycle: Synthesized in the liver -> intestines -> absorbed in the ileum ®portal circulation (recaptured by hepatocytes)
• Increases in fasting or postprandial serum bile acids concentrations are consistent with hepatic dysfunction, portosystemic shunting, or cholestasis.
• Serum bile acids are measured as preprandial sample (after withholding food for 12 hours) or by collection of paired preprandial and 2-hours postprandial samples
• For the diagnosis of hepatobiliary disease, the specificities of preprandial and postprandial bile acids concentrations are 100% (at values >20 mmol/L and >25 mmol/L, respectively).⁷

Important: Because the liver has a considerable reserve capacity, patients with liver disease can have normal liver function test results.

Canine Chronic Hepatitis (CCH)

• According to the WSAVA Standardization group, chronic hepatitis is characterized by hepatocellular apoptosis or necrosis, a variable mononuclear or mixed inflammatory infiltrate (mostly in the portal space), regeneration, and fibrosis.
• The proportion and distribution of these components vary widely (notably per breed).
• Histopathology is required to confirm the diagnostic and for prognostication.
• The activity of the disease is determined by the amount of inflammation and extent of hepatocellular apoptosis and necrosis.
• The stage of the disease, and the prognosis, may be determined by the extent and pattern of fibrosis and the possible presence of architectural distortion. Histochemical stains for connective tissue may be helpful in detecting the amount and pattern of fibrosis, particularly in early and mild disease.

Canine Copper-Associated hepatitis

• Diagnosis is based on histopathology as well as on hepatic copper evaluation. Semi-quantitative evaluation can be performed using special stains (rubeanic acid or rhodanine). Preferably, a quantitative evaluation is performed on unfixed frozen biopsy.
• Several breeds have been reported with an increased prevalence of chronic hepatitis, including (*indicates breeds with copper-associated hepatitis):
• Strong evidence: Bedlington terrier*, Labrador retriever*, American and English Cocker spaniel, Doberman pinscher*, West Highland white terrier, Dalmatian*, English Springer spaniel

Treatment of Chronic Hepatitis

• According to the severity/stage of the disease, consider nutraceutical hepatoprotectants (ex: ursodiol, SAM-e), protein restriction (should be limited to the maximum tolerated level to prevent signs of hepatic encephalopathy), GI protectant, +/- steroids or immunosuppressive agents, antifibrotic therapy.
• Anti-copper drug therapy is generally reserved for cases with documented primary or secondary excess copper accumulation. Specific treatment includes low-copper diet, copper chelator (penicillamine is typically used, trientine is an alternative), increased alimentary zinc.

References

1.  Comazzi S, Pieralisi C, Bertazzolo W. Haematological and biochemical abnormalities in canine blood: frequency and associations in 1022 samples. The Journal of Small Animal Practice. 2004;45(7):343–9.

2.  Ruaux CG, Carney PC, Suchodolski JS, Steiner JM. Estimates of biological variation in routinely measured biochemical analytes in clinically healthy dogs. Veterinary Clinical Pathology. 2012;41(4):541–7.

3.  Webster RL. Interpretation of serum alkaline phosphatase in dogs. NACV Clinician’s Brief - Consultant on call [Internet]. 2004:[9–12 pp.].

4.  Chapman SE, Hostutler RA. A laboratory diagnostic approach to hepatobiliary disease in small animals. Clinics in Laboratory Medicine. 2015;35(3):503–19.

5.  Gerritzen-Bruning MJ, van den Ingh TS, Rothuizen. Diagnostic value of fasting plasma ammonia and bile acid concentrations in the identification of portosystemic shunting in dogs. Journal of Veterinary Internal Medicine. 2006;20(1):13–9.

6.  Ruland K, Fischer A, Hartmann K. Sensitivity and specificity of fasting ammonia and serum bile acids in the diagnosis of portosystemic shunts in dogs and cats. Veterinary Clinical Pathology. 2010;39(1):57–64.

7.  Center SA, ManWarren T, Slater MR, Wilentz E. Evaluation of twelve-hour preprandial and two-hour postprandial serum bile acids concentrations for diagnosis of hepatobiliary disease in dogs. Journal of the American Veterinary Medical Association. 1991;199(2):217–26.