Iron, copper, and zinc are essential to numerous metalloenzymes and cellular processes involved with intermediary metabolism and xenobiotic detoxification in animals. The liver can serve as a repository for copper and iron, and excessive accumulation of these metals can contribute to tissue injury in various contexts. Importantly, copper and iron are considered transition metals because they fluctuate between ionic forms. In so doing, transition metals catalyze formation of reactive oxygen species from oxygen via the Fenton-driven Haber-Weiss reaction.1–3
The liver plays an important role in zinc metabolism because it constitutes an important pool of quickly exchanged zinc.4,5 Zinc is needed for proper liver function because it has important antioxidant, anti-inflammatory, and antiapoptotic properties and is essential for typical tissue replication and healing, immunoregulation, and ammonia detoxification. Tissue zinc insufficiency is recognized as a complication of liver injury or portosystemic shunting.4,5 Because a low liver zinc concentration can increase predisposition to certain hepatotoxins, may compromise tissue repair, reduces antioxidant protection, and can potentiate development of hyperammonemia and hepatic encephalopathy, measurement of zinc concentrations in liver tissue can be used when making decisions about therapeutic zinc supplementation.4,5
A liver biopsy specimen is estimated to constitute approximately 1/50,000 of the hepatic mass and is routinely interpreted as representing the health status of the entire organ.6 However, several studies in humans7–13 and 1 study in dogs and cats14 revealed the inaccuracy of needle-core biopsy for definitive diagnosis of liver disease and for therapeutic monitoring. Because of heterogeneity of lesion distribution, inadequate representation of acinar units, and collection of too few portal triads (< 11 to 15), needle-core biopsy specimens can be misleading and can yield erroneous results when analyzed.7–14 Specifically, histologic evaluation of needle-core biopsy specimens is reportedly inaccurate for detection of copper-storage hepatopathy and for sequential appraisal of treatment efficacy in humans with genetically confirmed Wilson's disease, even when copper-specific stains are applied.15,16 Further complicating analysis of small specimens is the heterogenous distribution of copper in liver tissue in humans15–20 and other animals.21–24
Hepatic copper concentrations in dogs (< 400 μg/g [ppm] of dry-weight liver tissue) are typically substantially higher than in most other animal species.25–27 Humans, for example, have a typical hepatic copper concentration of only 10 to 30 ppm.27,28 Hepatic copper concentrations > 2,000 ppm of dry-weight liver tissue in dogs are often associated with progressive liver injury. However, some dogs tolerate extraordinary increases in hepatic copper concentrations without developing liver damage.27,29
Hepatic iron concentrations in clinically normal dogs range from 400 to 1,200 ppm of dry-weight liver tissue. A high iron concentration is common in dogs with necroinflammatory liver disease. It is probable that this high iron concentration contributes to or makes tissues vulnerable to injury as the liver engages in its usual sentinel functions, protecting the systemic circulation from the toxins and enteric pathogens delivered in the portal circulation.1,3,30,31 Retention of transition metals in the liver may contribute to the considerable glutathione depletion that has been detected in dogs with spontaneous necroinflammatory and cholestatic liver injury.32
It is unknown whether measurement of metal concentrations in liver biopsy specimens from dogs is influenced by specimen size or lesion heterogeneity. In humans, a minimum of 3 to 5 mg of dry-weight tissue (equivalent to a minimum of approx 6 to 10 mg of wet tissue) is a standard requirement when performing metal analyses of needle-core biopsy specimens.15,33,34 Smaller specimens are considered unsatisfactory for determination of copper concentration because the distribution of copper within the tissue may be inhomogenous, particularly in architecturally remodeled liver containing regenerative nodules and extensive fibrosis.15 Regional variability in copper concentrations has been confirmed in many animals (eg, humans, pigs, copper-fed rats, neonatal calves, and weanling rats).15–24 In humans, variability in copper concentrations between specimens of the same liver has been detected in neonates, adults, and patients with cirrhosis or other necroinflammatory liver disorders.15–20 Such variability has the potential to complicate diagnostic conclusions and therapeutic recommendations, particularly when stained needle-core biopsy specimens are used for subjective assessments or when specimens are submitted for quantitative metal analyses.
In veterinary medicine, it is increasingly common for clinicians to request an analysis of metal concentrations in formalin-fixed, paraffin-embedded, xyleneextracted tissue after receipt of histologic descriptions suggesting that excessive hepatic copper or iron stores have contributed to liver injury. There is no information confirming that such measurements correlate well with measurements obtained from fresh, unfixed tissue. The purpose of the study reported here was to determine the influence of specimen size on metal concentrations in liver tissue as measured by use of atomic absorption spectrometry, the variability associated with measurement of metal concentrations in the same liver tissue when biopsy specimens from the same liver lobe are analyzed on different days, and the validity of measurements of copper, iron, and zinc concentrations in formalin-fixed, paraffin-embedded, xylene-extracted liver specimens.
Coefficient of variation
Bard Urological Division, Covington, Ga.
Braselton WE, Slanker MR, Stuart KJ, et al. Comparison of element concentrations determined in fresh, formalin fixed and paraffin embedded tissue samples (abstr), in Proceedings. 40th Meet Am Assoc Vet Lab Diagn 1997;74.
Varian AA-1275, Varian Inc, Palo Alto, Calif.
Certified standardizing stock solutions (ferric nitrate, zinc oxide, copper nitrate) in nitric acid, 1,000 ppm ± 1%, Fisher Scientific, Pittsburgh, Pa.
Diagnostic Laboratory, College of Veterinary Medicine, University of Colorado, Fort Collins, Colo.
Statistix, version 9.0, Analytical Software, Tallahassee, Fla.
Letelier ME, Lepe AM, Faúndez M, et al.Possible mechanisms underlying copper-induced damage in biological membranes leading to cellular toxicity. Chem Biol Interact 2005;151:71–82.
Ramm GA, Ruddell RG. Hepatotoxicity of iron overload: mechanisms of iron-induced hepatic fibrogenesis. Semin Liver Dis 2005;25:433–449.
Regev A, Berho M, Jeffers LJ, et al. Sampling error and intraobserver variation in liver biopsy in patients with chronic HCV infection. Am J Gastroenterol 2002;97:2614–2618.
Holund B, Poulsen H, Schlichting P. Reproducibility of liver biopsy diagnosis in relation to the size of the specimen. Scand J Gastroenterol 1980;15:329–335.
Schlichting P, Holund B, Poulsen H. Liver biopsy in chronic aggressive hepatitis. Diagnostic reproducibility in relation to size of specimen. Scand J Gastroenterol 1983;18:27–32.
Poniachik J, Bernstein DE, Reddy KR, et al. The role of laparoscopy in the diagnosis of cirrhosis. Gastrointest Endosc 1996;43:568–571.
Cholongitas E, Senzolo M, Standish R, et al. A systematic review of the quality of liver biopsy specimens. Am J Clin Pathol 2006;125:710–721.
Van Leeuwen DJ, Balabaud C, Crawford JM, et al. A clinical and histopathologic perspective on evolving noninvasive and invasive alternatives for liver biopsy. Clin Gastroenterol Hepatol 2008;6:491–496.
Cole TL, Center SA, Flood SN, et al. Diagnostic comparison of needle and wedge biopsy specimens of the liver in dogs and cats. J Am Vet Med Assoc 2002;220:1483–1490.
Ferenci P, Steindl-Munda P, Vogel W. Diagnostic value of quantitative hepatic copper determination in patients with Wilson's disease. Clin Gastroenterol Hepatol 2005;3:811–818.
Diaz G, Faa G, Fami AMG, et al. Copper distribution within and between newborn livers. J Trace Elem Electrolytes Health Dis 1990;4:61–64.
Milman N, Laursen G, Podenphant J, et al. Trace elements in normal and cirrhotic human liver tissue. Iron, copper, zinc, selenium, manganese, titanium and lead measured by X-ray fluorescence spectroscopy. Liver 1986;6:111–117.
Faa G, Diaz G, Farci AMG, et al. Variability of copper levels in biopsy tissue from a cirrhotic liver. J Trace Elem Electrolytes Health Dis 1990;4:49–50.
Cassidy J, Eva JK. The variations in the concentrations of copper and iron within and between the lobes of pig's liver. Proc Nutr Soc 1958;17:30.
Howell JS. Histochemical demonstration of copper in copperfed rats and in hepatocellular degeneration. J Pathol Bacteriol 1959;77:473–483.
Su LC, Owen CA, Zollman PE, et al. A defect of biliary excretion of copper in copper-laden Bedlington terriers. Am J Physiol 1982;343:G231–G236.
Thornburg LP, Rottinghaus G, McGowan M, et al. Hepatic copper concentrations in purebred and mixed-breed dogs. Vet Pathol 1990;27:81–88.
Linder MC. Introduction and overview of copper as an element. In: Linder MC, ed. Biochemistry of copper. New York: Plenum Press, 1991;1–15.
Thornburg LP, Rottinghaus G, Dennis G, et al. The relationship between hepatic copper content and morphologic changes in the liver of West Highland White Terriers. Vet Pathol 1996;33:656–661.
Center SA. Metabolic, antioxidant, nutraceutical, probiotic, and herbal therapies relating to the management of hepatobiliary disorders. Vet Clin North Am Small Anim Pract 2004;34:67–172.
Center SA, Warner KL, Erb HN. Liver glutathione concentrations in dogs and cats with naturally occurring liver disease. Am J Vet Res 2002;63:1187–1197.
Association of Official Analytical Chemists (AOAC). In: Helrich K, ed. Official methods of analysis. 15th ed. Arlington, Va: AOAC, 1990.
Thornburg LP, Beissenherz M, Dolan M, et al. Histochemical demonstration of copper and copper-associated protein in the canine liver. Vet Pathol 1985;22:327–332.
Bischoff K, Lamm C, Erb HN, et al. The effects of formalin fixation and tissue embedding of bovine liver on copper, iron, and zinc analysis. J Vet Diagn Invest 2008;20:220–224.
Nooijen JL, van den Hamer CJA, Houtman JPW, et al.Possible errors in sampling percutaneous liver biopsies for determination of trace element status: application to patients with primary biliary cirrhosis. Clin Chim Acta 1981;113:335–338.
Goldfischer S, Popper H, Sternlieb I. The significance of variations in the distribution of copper in liver disease. Am J Pathol 1980;99:715–730.
Guido M, Colloredo G, Fassan M, et al. Clinical practice and ideal liver biopsy sampling standards: not just a matter of centimeters. J Hepatol 2006;44:818–826.
Schultheiss PC, Bedwell CL, Hamar DW. Canine liver iron, copper, and zinc concentrations and association with histologic lesions. J Vet Diagn Invest 2002;14:396–402.
Pietrangelo A, Gualdi R, Casalgrandi G, et al. Molecular and cellular aspects of iron-induced hepatic cirrhosis in rodents. J Clin Invest 1995;95:1824–1831.
Jain S, Scheuer PJ, Archer B, et al. Histological demonstration of copper and copper-associated protein in chronic liver disease. J Clin Patho1 1978;31:784–790.
Theodossi A, Skene AM, Portmann B, et al. Observer variation in assessment of liver biopsies including analysis by Kappa statistics. Gastroenterology 1980;79:232–241.
Thornburg LP, Rottinghaus G, Gage H. Chronic liver disease associated with high hepatic copper concentration in a dog. J Am Vet Med Assoc 1986;188:1190–1191.
Webb CB, Twedt DC, Meyer DJ. Copper-associated liver disease in Dalmatians: a review of 10 dogs (1998–2001). J Vet Intern Med 2002;16:665–668.
Hoffmann G, van den Ingh TS, Bode P, et al.Copper-associated chronic hepatitis in Labrador Retrievers. J Vet Intern Med 2006;20:856–861.