Canine models of copper toxicosis for understanding mammalian copper metabolism.

Hereditary forms of copper toxicosis exist in man and dogs. In man, Wilson's disease is the best studied disorder of copper overload, resulting from mutations in the gene coding for the copper transporter ATP7B. Forms of copper toxicosis for which no causal gene is known yet are recognized as well, often in young children. Although advances have been made in unraveling the genetic background of disorders of copper metabolism in man, many questions regarding disease mechanisms and copper homeostasis remain unanswered. Genetic studies in the Bedlington terrier, a dog breed affected with copper toxicosis, identified COMMD1, a gene that was previously unknown to be involved in copper metabolism. Besides the Bedlington terrier, a number of other dog breeds suffer from hereditary copper toxicosis and show similar phenotypes to humans with copper storage disorders. Unlike the heterogeneity of most human populations, the genetic structure within a purebred dog population is homogeneous, which is advantageous for unraveling the molecular genetics of complex diseases. This article reviews the work that has been done on the Bedlington terrier, summarizes what was learned from studies into COMMD1 function, describes hereditary copper toxicosis phenotypes in other dog breeds, and discusses the opportunities for genome-wide association studies on copper toxicosis in the dog to contribute to the understanding of mammalian copper metabolism and copper metabolism disorders in man.

The Linoleic Acid Content of the Stratum Corneum of Ichthyotic Golden Retriever Dogs Is Reduced as Compared to Healthy Dogs and a Significant Part Is Oxidized in Both Free and Esterified Forms.

Golden Retrievers may suffer from Pnpl1-related inherited ichthyosis. Our study shows that in the stratum corneum (SC) of ichthyotic dogs, linoleic acid (LA) is also present in the form of 9-keto-octadecadienoic acid (9-KODE) instead of the acylacid form as in normal dogs. The fatty acids purified from SC strips (LA, acylacids) were characterized by liquid chromatography-tandem mass spectrometry (LC-MS) and atmospheric pressure chemical ionization (APCI). Electrospray ionization (ESI) and MS2(MS/MS Tandem mass spectrum/spectra)/M3 (MS/MS/MS Tandem mass spectrum/spectra) fragmentation indicated the positions of the double bonds in 9-KODE. We showed that ichthyotic dogs have a threefold lower LA content in the form of acylacids. The MS2 fragmentation of acyl acids showed in some peaks the presenceof an ion at the m/z 279, instead of an ion at m/z 293 which is characteristic of LA. The detected variant was identified upon MS3 fragmentation as 9-keto-octadecadienoic acid (9-KODE), and the level of this keto-derivative was increased in ichthyotic dogs. We showed by the APCI that such keto forms of LA are produced from hydroperoxy-octadecadienoic acids (HpODE) upon dehydration. In conclusion, the free form of 9-KODE was detected in ichthyotic SC up to fivefold as compared to unaffected dogs, and analyses by HPLC (High performance liquid chromatography) and ESI-MS (Electrospray Ionization-Mass Spectrometry) indicated its production via dehydration of native 9-HpODE.

Association of the canine ATP7A and ATP7B with hepatic copper accumulation in Dobermann dogs.

BACKGROUND: Hepatic copper accumulation causes chronic hepatitis in dogs. Mutations in the copper transporters ATP7A and ATP7B were, respectively, associated with attenuation and enhancement of hepatic copper concentrations in Labrador Retrievers. There is a predisposition of Dobermanns to hepatitis with increased hepatic copper concentrations. OBJECTIVES: To investigate whether the ATP7A:c.980C>T and ATP7B:c.4358G>A mutations identified in Labrador Retrievers were associated with hepatic copper concentrations in Dobermanns. ANIMALS: Dobermanns from the Netherlands (n = 122) and the United States (n = 78). METHODS: In this retrospective study, mutations in ATP7A and ATP7B were investigated as risk factors for hepatic copper accumulation in Dobermanns. Liver biopsies of 200 Dobermanns were evaluated by histochemical copper staining, quantitative copper measurement, or both modalities. ATP7A and ATP7B genotypes were obtained by Kompetitive Allele Specific PCR. A linear regression model was used to investigate an association between genotype and hepatic copper concentrations. RESULTS: The ATP7A:c.980C>T was identified in both Dutch (2 heterozygous individuals) and American Dobermanns. In the American cohort, the minor allele frequency of the mutation was low (.081) and a possible effect on hepatic copper concentrations could not be established from this data set. A significant association of the ATP7B:c.4358G>A variant with increased hepatic copper concentrations in Dobermanns was observed. CONCLUSIONS AND CLINICAL IMPORTANCE: The ATP7B:c.4358G>A variant could be a contributor to hepatic copper accumulation underlying the risk of development of copper-associated hepatitis in breeds other than the Labrador Retriever.

Linoleate-enriched diet increases both linoleic acid esterified to omega hydroxy very long chain fatty acids and free ceramides of canine stratum corneum without effect on protein-bound ceramides and skin barrier function.

Few studies have investigated the influence of increased amounts of dietary linoleic acid on the epidermal lipid biochemistry and TEWL in healthy subject. The influence of dietary linoleic acid on canine stratum corneum (SC) lipids was studied by feeding two groups of five dogs differential amounts of linoleic acid (LA) for three months. SC was harvested by tape stripping and lipids were analyzed by thin-layer chromatography and mass spectrometry. The dogs that were fed the higher concentration of LA showed high increases in the contents of both linoleic acid and free ceramides in the SC, whereas the protein-bound ceramide content was unchanged. Acylacids that represent the esterified form of linoleic acid in omega hydroxy very long chain fatty acids (ω-OH VLCFA) accounted for most of the elevation of LA, whereas the concentration of the free form was not significantly changed. Corroborating the absence of change in the protein-bound ceramides content of healthy dogs SC, TEWL was nearly unaffected by the linoleic acid-enriched diet.

Gene expression patterns in the progression of canine copper-associated chronic hepatitis.

Copper is an essential trace element, but can become toxic when present in abundance. The severe effects of copper-metabolism imbalance are illustrated by the inherited disorders Wilson disease and Menkes disease. The Labrador retriever dog breed is a novel non-rodent model for copper-storage disorders carrying mutations in genes known to be involved in copper transport. Besides disease initiation and progression of copper accumulation, the molecular mechanisms and pathways involved in progression towards copper-associated chronic hepatitis still remain unclear. Using expression levels of targeted candidate genes as well as transcriptome micro-arrays in liver tissue of Labrador retrievers in different stages of copper-associated hepatitis, pathways involved in progression of the disease were studied. At the initial phase of increased hepatic copper levels, transcriptomic alterations in livers mainly revealed enrichment for cell adhesion, developmental, inflammatory, and cytoskeleton pathways. Upregulation of targeted MT1A and COMMD1 mRNA shows the liver's first response to rising intrahepatic copper concentrations. In livers with copper-associated hepatitis mainly an activation of inflammatory pathways is detected. Once the hepatitis is in the chronic stage, transcriptional differences are found in cell adhesion adaptations and cytoskeleton remodelling. In view of the high similarities in copper-associated hepatopathies between men and dog extrapolation of these dog data into human biomedicine seems feasible.

The Menkes and Wilson disease genes counteract in copper toxicosis in Labrador retrievers: a new canine model for copper-metabolism disorders.

The deleterious effects of a disrupted copper metabolism are illustrated by hereditary diseases caused by mutations in the genes coding for the copper transporters ATP7A and ATP7B. Menkes disease, involving ATP7A, is a fatal neurodegenerative disorder of copper deficiency. Mutations in ATP7B lead to Wilson disease, which is characterized by a predominantly hepatic copper accumulation. The low incidence and the phenotypic variability of human copper toxicosis hamper identification of causal genes or modifier genes involved in the disease pathogenesis. The Labrador retriever was recently characterized as a new canine model for copper toxicosis. Purebred dogs have reduced genetic variability, which facilitates identification of genes involved in complex heritable traits that might influence phenotype in both humans and dogs. We performed a genome-wide association study in 235 Labrador retrievers and identified two chromosome regions containing ATP7A and ATP7B that were associated with variation in hepatic copper levels. DNA sequence analysis identified missense mutations in each gene. The amino acid substitution ATP7B:p.Arg1453Gln was associated with copper accumulation, whereas the amino acid substitution ATP7A:p.Thr327Ile partly protected against copper accumulation. Confocal microscopy indicated that aberrant copper metabolism upon expression of the ATP7B variant occurred because of mis-localization of the protein in the endoplasmic reticulum. Dermal fibroblasts derived from ATP7A:p.Thr327Ile dogs showed copper accumulation and delayed excretion. We identified the Labrador retriever as the first natural, non-rodent model for ATP7B-associated copper toxicosis. Attenuation of copper accumulation by the ATP7A mutation sheds an interesting light on the interplay of copper transporters in body copper homeostasis and warrants a thorough investigation of ATP7A as a modifier gene in copper-metabolism disorders. The identification of two new functional variants in ATP7A and ATP7B contributes to the biological understanding of protein function, with relevance for future development of therapy.

Uterine glands provide histiotrophic nutrition for the human fetus during the first trimester of pregnancy.

Providing adequate nutrition to the fetus is key to a successful pregnancy. The interstitial form of implantation displayed by the human blastocyst is generally associated with early onset of maternal blood flow to the developing placenta, and hence hemotrophic exchange. However, the recent finding that the maternal intraplacental circulation is not fully established until the third month of gestation suggests that human fetal nutrition may be initially histiotrophic. We therefore investigated activity of the uterine glands during early pregnancy. We demonstrate here that these glands remain active until at least wk 10 of pregnancy, and that their secretions are delivered freely into the placental intervillous space. We also demonstrate phagocytic uptake by the placental syncytiotrophoblast of two glycoproteins, the mucin MUC-1 and glycodelin A, synthesized in the maternal glands. Glycodelin was also detected within the epithelium of the secondary yolk sac lining the exocoelomic cavity, indicating that the yolk sac may play an important role in nutrient exchange before vascularisation of the chorionic villi. Our findings demonstrate that the uterine glands are an important source of nutrients during organogenesis, when metabolism is essentially anaerobic.

Nutritional management of inherited copper-associated hepatitis in the Labrador retriever.

Canine hereditary copper-associated hepatitis is characterized by gradual hepatic copper accumulation eventually leading to liver cirrhosis. Therapy is aimed at creating a negative copper balance with metal chelators, of which D-penicillamine is the most commonly used. D-penicillamine often causes gastro-intestinal side effects and life-long continuous therapy may lead to a deficiency of copper and zinc. The aim of the current study was to investigate the effect of a low-copper, high-zinc diet as an alternative to continuous D-penicillamine treatment for the long-term management of canine copper-associated hepatitis. Sixteen affected Labrador retrievers were followed for a median time period of 19.1 months (range, 5.9-39 months) after being effectively treated with D-penicillamine. The dogs were maintained on a diet containing 1.3±0.3 mg copper/1000 kcal and 64.3±5.9 mg zinc/1000 kcal. Liver biopsies were taken every 6 months for histological evaluation and copper determination. Plasma alanine aminotransferase (ALT) and alkaline phosphatase, as well as serum albumin were determined. Dietary treatment alone was sufficient to maintain hepatic copper concentration below 800 mg/kg dry weight liver in 12 dogs during the study period. Four dogs needed re-treatment with D-penicillamine. ALT activity and albumin concentration were not associated with hepatic copper concentration, but showed a significant association with the stage and grade of hepatitis respectively. In conclusion, a low-copper, high-zinc diet can be a valuable alternative to continuous d-penicillamine administration for long-term management of dogs with copper-associated hepatitis. The copper re-accumulation rate of an individual dog should be considered in the design of a long-term management protocol and in determining re-biopsy intervals.

D-penicillamine treatment of copper-associated hepatitis in Labrador retrievers.

d-penicillamine is effectively used in the lifelong treatment of copper toxicosis in Bedlington terriers and Wilson's disease in humans. A complex form of copper-associated hepatitis has recently been characterized in the Labrador retriever. The aims of this study were to evaluate the effectiveness of d-penicillamine treatment for copper-associated hepatitis in this breed, to study the effects on hepatic copper, iron and zinc concentrations, and to evaluate parameters to predict optimal duration of treatment. Forty-three client owned Labrador retrievers that were diagnosed with increased hepatic copper were treated with d-penicillamine and underwent at least one follow-up examination including a liver biopsy for histopathological scoring of inflammatory lesions. Hepatic copper, iron and zinc concentrations were determined in the initial and follow-up biopsies by instrumental neutron activation analysis. The influence of initial hepatic copper concentration, sex, age, d-penicillamine formulation and the occurrence of side effects were investigated for their influence on hepatic copper concentration after a certain period of treatment by generalized mixed modelling. d-penicillamine proved to be effective in reducing hepatic copper concentration and associated inflammatory lesions. Parameters derived from the model can be used to estimate the necessary duration of d-penicillamine treatment for Labrador retrievers with increased hepatic copper concentration. Continuous, lifelong d-penicillamine treatment is not recommended in this breed, as there may be a risk for hepatic copper and zinc deficiency.

Assessment of methods of evaluating sarcopenia in old dogs.

OBJECTIVE: To evaluate clinically applicable methods of assessing lean body mass in dogs and compare muscle mass and inflammatory markers in healthy young and old dogs. ANIMALS: 9 healthy young (1 to 5 years old) and 10 old (> 8 years old) Labrador Retrievers with a body condition score of 5 to 6 of 9. PROCEDURES: Radiography of the thoracolumbar region was performed for measurement of epaxial muscle height at the level of T13-L1. Computed tomographic images were obtained for the measurement of the epaxial and temporal muscles. Ultrasonography also was performed for regional muscle measurements at these same sites and the quadriceps muscle. Serum C-reactive protein, insulin-like growth factor-1, and tumor necrosis factor-α concentrations also were measured, and dogs' activity for 14 days was assessed with an activity monitor. RESULTS: Mean epaxial muscle area measured by ultrasonography was significantly lower in the old group, compared with the young group, whereas epaxial muscle area measured by CT was only significantly lower in the old group after normalization for vertebral height. Neither temporal and quadriceps muscle measurements nor serum C-reactive protein or insulin-like growth factor-1 concentrations were significantly different between age groups. Tumor necrosis factor-α concentrations were undetectable in all dogs. CONCLUSIONS AND CLINICAL RELEVANCE: This study documented reduced epaxial muscle area in healthy old Labrador Retrievers, consistent with the syndrome of sarcopenia. Ultrasonography and CT were feasible methods of measuring epaxial muscle area, but much additional research is required to assess this method. A better understanding of underlying mechanisms of sarcopenia as well as methods for slowing progression is needed.

Dietary constituents are able to play a beneficial role in canine epidermal barrier function.

Epidermal barrier function is a critical attribute of mammalian skin. The barrier is responsible for preventing skin-associated pathologies through controlling egress of water and preventing ingress of environmental agents. Maintaining the quality and integrity of the epidermal barrier is therefore of considerable importance. Structurally, the barrier is composed of two main parts, the corneocytes and the intercellular lamellar lipid. The epidermal lamellar lipid comprises mainly ceramides, sterols and fatty acids. Twenty-seven nutritional components were screened for their ability to upregulate epidermal lipid synthesis. Seven of the 27 nutritional components (pantothenate, choline, nicotinamide, histidine, proline, pyridoxine and inositol) were subsequently retested using an in vitro transepidermal diffusion experimental model, providing a functional assessment of barrier properties. Ultimately, the best performing five nutrients were fed to dogs at supplemented concentrations in a 12-week feeding study. Barrier function was measured using transepidermal water loss (TEWL). It was found that a combination of pantothenate, choline, nicotinamide, histidine and inositol, when fed at supplemented concentrations, was able to significantly reduce TEWL in dogs after 9 weeks.