Emerging copper-associated hepatopathies in dogs (Proceedings)

Chronic hepatitis associated with abnormal accumulation of copper in liver cells is emerging as an important form of chronic liver disease in dogs. As with other forms of chronic hepatitis, copper-associated hepatitis is characterized by hepatocellular necrosis (or apoptosis), inflammation, regeneration, fibrosis, and progression to cirrhosis. Breed-specific copper-associated hepatitis has been described in the Bedlington Terrier, West Highland White Terrier, Skye Terrier, Doberman Pinscher, Dalmatian, and Labrador Retriever, but many other breeds can also have increased hepatic copper.

Copper is an essential trace element in the diet required for normal metabolism, but in excessive concentrations copper is toxic to cells such as hepatocytes through oxidative injury. The primary source of copper is through dietary intake, and 40% to 60% of ingested copper is absorbed in the proximal intestine and transported to the liver. Copper is stored in the lysosomes of liver cells, and excess copper is excreted from the body in bile. The minimum copper level in dog food recommended by AAFCO is 7.3 ppm (mg/kg, DW); most commercial dog foods contain 12 to 16 ppm (mg/kg, DW).

Hepatic copper accumulation can be primary or secondary, and it can result from increased uptake, defective metabolism, or impaired biliary excretion. Primary copper hepatopathy is an inherited copper storage disease caused by an underlying genetic defect in copper metabolism, which results in the progressive lifelong accumulation of hepatic copper and culminates in liver cell death, chronic hepatitis, and cirrhosis. Secondary accumulation of hepatic copper is a consequence of acquired liver diseases that produce chronic cholestasis and decreased biliary excretion of copper. The copper accumulation in the primary (genetic) form is centrilobular (zone 3), and in the secondary (cholestatic) form the copper accumulation is periportal.

Hepatic copper can be measured quantitatively on liver biopsies obtained by 16-gauge biopsy needle, laparoscopy, or surgery. Liver specimens should be submitted frozen for mineral analysis in a copper-free vial (or a red-top tube with cellophane covering the rubber cap) to a lab capable of copper analysis (e.g., Michigan State University; Colorado State University). Normal liver copper is less than 400 ppm (ppm = μg/g = mg/kg), dry weight (DW). Hepatic copper levels exceeding 2,000 ppm are consistently hepatotoxic by oxidant injury. Liver damage is seen in some dogs (especially non-Bedlington breeds) at lower copper levels of 800 to 1,200 ppm. Copper stains (rhodanine; rubeanic acid) can be used for semiquantitative assessment of copper in liver biopsies and cytologies, such as ultrasound-guided fine-needle liver aspirates (FNA). With standard cytology and H&E stains, copper appears as golden brown refractile granules.

Bedlington terrier

Copper storage disease in Bedlingtons is autosomal recessive with homozygous dogs developing the clinically apparent liver disease. The defect is an exon 2 deletion in the gene encoding copper transport protein COMMD1 (formerly MURR1). This defect interferes with biliary copper excretion, so hepatic copper accumulates progressively over the lifetime of the dog. Affected Bedlingtons have copper levels of 1,000 to 12,000 ppm (average, 6,000 ppm), and hepatotoxicity is consistently observed when copper exceeds 2,000 ppm. During the accumulation phase, which can last for years, clinical signs and laboratory abnormalities are absent. Eventually the serum ALT increases, then chronic hepatitis and cirrhosis become evident. The severity of liver disease tends to reflect the copper level in the liver. Nonspecific clinical signs include inppetance, lethargy, weight loss, and vomiting. With progression to cirrhosis, signs of jaundice, ascites, coagulopathy, and hepatic encephalopathy may develop. Occasionally acute hepatic necrosis releases copper into the circulation causing hemolytic anemia.

DNA testing for Bedlington terriers is available from VetGen (www.vetgen.com). This assay evaluates a linkage-based DNA marker (C04107) for the defective gene. The test can identify normal, affected, and carrier dogs with 90% accuracy. False negatives in some dogs might indicate a variant genetic defect. Liver biopsy is still recommended as the “gold standard” for breeding dogs. With early diagnosis and treatment (chelators, zinc, diet) most affected Bedlingtons can lead a normal life.

Doberman pinscher

Middle-aged Dobermans are at increased risk of developing chronic hepatitis and cirrhosis associated with hepatic copper accumulation. The pathogenesis is unclear, but a genetic basis is supsected. Females are affected more than males. Clinical cases are often diagnosed in the advanced stages of hepatic failure with variable signs of weight loss, ascites, jaundice, bleeding, and hepatic encephalopathy. Laboratory findings include increased liver enzymes, hyperbilirubinemia, hypoalbuminemia, hyperammonemia, and coagulopathy. Liver biopsy reveals portal inflammation (lymphocytic-plasmacytic), piecemeal and bridging necrosis, bile duct hyperplasia, and portal fibrosis. Hepatic copper concentrations are typically 1000 to 2000 ppm, but up to 4,700 ppm has been seen. It has been debated whether the copper accumulation in Doberman hepatitis is a primary defect or secondary to cholestasis. Studies in Dobermans with early subclinical hepatitis have shown that centrilobular copper accumulation precedes the development of cholestasis and severe inflammatory lesions, which supports a primary copper retention disorder. Other studies have suggested hepatic copper accumulates secondary to chronic immune-mediated hepatitis in Dobermans.

Chelation therapy of Dobermans with early subclinical hepatitis appears to prevent progression of the disease; thus, it is recommended that Dobermans over 1 year of age be screened periodically for increased serum ALT, and that dogs with persistent ALT elevation have a liver biopsy and quantitative copper analysis so treatment can initiated early. Dobermans presenting with advanced hepatitis or cirrhosis have a guarded to poor prognosis. In addition to de-coppering the liver in Dobermans with clinical hepatitis, other treatment considerations should include anti-inflammatory, immunosuppressive, and hepatoprotective strategies.

West highland white terrier (WHWT)

A familial copper-associated chronic hepatitis is recognized in the WHWT. Decreased biliary excretion of copper has been demonstrated in conjunction with centrilobular copper accumulation during the first year of life. Liver biopsies can show multifocal centrilobular hepatitis, subacute bridging necrosis, massive necrosis, and cirrhosis. Hepatic copper levels are usually < 2,000 ppm and rarely exceed 3,500 ppm. The relationship of hepatic copper to chronic hepatitis in the WHWT is unclear and the mode of inheritance is unknown.

Labrador retriever

Labradors are at increased risk for chronic hepatitis associated with increased levels of hepatic copper (mean 3,369 ppm; range 2,375 to 4,972). Most labs present at 7 to 9 years of age (range, 2.5 to 14 yr). Increased hepatic copper has been found in asymptomatic dogs genetically related to labs with overt hepatitis, suggesting a genetic basis for this disease. Treatment with penicillamine appears to be effective for decreasing hepatic copper and inflammation. Some dogs also respond to immunosuppressive therapy (prednisone, azathioprine) and hepatoprotectives (SAMe, milk thistle, ursodiol).

Dalmatian

Dalmations have been reported with acute hepatic necrosis, chronic hepatitis, and cirrhosis associated with increased hepatic copper (mean, 3,197 ppm; range 754 to 8,390 ppm). Liver biopsies showed piecemeal necrosis, inflammation (lymphocytes, neutrophils) and bridging fibrosis. Cholestasis is not a prominent feature in these cases, suggesting copper accumulation is due to a primary metabolic defect rather than secondary to cholestasis. In addition, two of the reported cases were genetically related. Most dogs present initially for acute GI signs (anorexia, vomiting, diarrhea) with markedly increased serum ALT and lesser increase in ALP. Advanced cases develop hyperbilirubinemia, hypoalbuminemia, and sometimes renal glucosuria and proteinuria. Disease progression was rapid.

Skye terrier

Chronic hepatitis and cirrhosis associated with hepatic copper accumulation (800 to 2,200 ppm) has been described in genetically related Skye Terriers. Cholestasis and hepatic inflammation precede the copper accumulation, suggesting this might represent secondary copper retention from cholestasis.

Treatment of copper-associated hepatitis

Copper-associated hepatitis is treated by copper chelator therapy (penicillamine), decreased intake and intestinal absorption of copper, and antioxidant therapy.

Chelator therapy

Dogs with copper-associated hepatitis should be treated with the copper chelator penicillamine (10 to 15 mg/kg PO q12h), especially if hepatic copper concentration exceeds 1,500 ppm. In addition to its copper-lowering effects, penicillamine has anti-inflammatory, antifibrotic, and immunomodulation effects. Trientine (15 mg/kg PO q12h) is a comparable alternative drug for chelation. Chelators are most effective if given on an empty stomach. Side effects of anorexia, nausea, and vomiting are common, which may necessitate giving penicillamine with a small amount of food. Owners should be informed to wear gloves when handling penicillamine and trientine because they are considered to be teratogenic.

Depending on the severity of the hepatic copper accumulation, treatment for several months or even years may be required to completely “de-copper” the liver. Substantial individual variation in the response to chelators has been observed. One study in Bedlingtons found that chelation decreased copper by an average of 1,500 ppm after 6 months. A group of Dobermans treated for 4 months with penicillamine had an average decrease of 600 ppm. A study in Labradors reported chelation to be successful in 3 to 6 months. The decision to discontinue chelator therapy ideally should be based on a followup liver biopsy for quantitative copper analysis, or on ultrasound-guided FNA cytology stained with a copper stain.

Therapy to decrease GI absorption of copper

The GI absorption of copper can be decreased by feeding a copper-restricted diet and administering oral zinc. These measures are considered adjuncts to chelation and are not effective alone for removing large concentrations of copper from the liver. Commercial liver diets are formulated with low copper levels (3 to 5 ppm) and added zinc (to decrease copper absorption). In addition, mineral supplements and foods high in copper should be avoided (e.g., eggs, liver, shellfish, organ meats, nuts, beans, mushrooms, cereals, dog treats). Copper-free distilled water can be used in place of tap water.

Zinc acetate or gluconate (100 mg elemental zinc, PO q12h for 3 months; then 50 mg PO q12h) can be used as maintenance therapy after copper chelation, or as initial therapy in lieu of chelation when hepatic copper levels are only mildly increased. Zinc induces copper-binding protein metallothionein, which preferentially binds copper and prevents its absorption, but zinc must be given for a minimum of 3 months to have this effect. Ideally zinc administration should be separated from meals by at least one hour and not combined with chelator therapy. Oral zinc given concurrently with penicillamine will be chelated and reduce the effectiveness of both drugs.

Antioxidant therapy

Copper accumulation, inflammation, and cholestasis all contribute to oxidant injury and death of hepatocytes. Increased hepatic iron levels seen in copper-associated hepatitis may also contribute to oxidant injury. Therefore hepatoprotective antioxidant therapy with S-adenosylmethionine (SAMe; 20 mg/kg/day) and/or vitamin E (10-15 IU/kg/day) are recommended. Other cytoprotective agents such as silymarin (milk thistle) and ursodiol may also be beneficial.

Corticosteroids

Corticosteroids are not generally recommended for treatment of hepatitis associated with primary hepatic copper accumulation, such as in Bedlingtons. However, in Dobermans and possibly some of the other breeds with copper-associated hepatitis, it is unclear whether the hepatic copper accumulation is causal or secondary to an underlying idiopathic (immune-mediated) hepatitis. If immune-mediated idiopathic hepatitis is suspected, corticosteroid therapy (prednisone or prednisolone, 1 to 2 mg/kg q24h, tapered to 0.5 to 1 mg/kg q24-48h) may have beneficial anti-inflammatory, immune-modulating, and anti-fibrotic effects. Prednisone can also be combined with an immunosuppressive, such as azathioprine in this situation.

References

Available on request